Devices, Methods, and Systems for Fluorescence-Based Imaging and Collection of Data for Food Safety Purposes

ABSTRACT

A method of screening for contamination during food production is disclosed. The method includes applying an exogenous, bacteria-specific contrast agent to a surface to be screened, wherein the surface to be screened is one or more of a food product, a food-preparation surface, a food-handling surface, and a food-equipment surface. The surface is illuminated with excitation light emitted by at least one light excitation light source of a handheld device and optical signals responsive to illumination of the surface are filtered with at least one optical filter of the handheld device. Bacterial fluorescence data regarding the illuminated surface is collected with an image sensor of the handheld device and, in real-time, based on the collected bacterial fluorescence data, the presence and/or location of bacteria on the illuminated surface is determined

This is application is a continuation application of U.S. applicationSer. No. 14/719,493, filed May 22, 2015, which is a continuationapplication of U.S. application Ser. No. 12/992,040, filed on Feb. 7,2011, now U.S. Pat. No. 9,042,967, which is a national stage applicationof PCT/CA2009/000680, filed internationally on May 20, 2009, whichclaims benefit to U.S. Provisional Application No. 61/054,780, filed May20, 2008, the entire content of each of which is incorporated byreference herein.

TECHNICAL FIELD

A device and method for fluorescence-based imaging and monitoring isdisclosed. In particular, the device and method may be suitable formonitoring biochemical and/or biological and non-biological substances,such as for the detection of bacteria.

BACKGROUND

Wound care is a major clinical challenge. Healing and chronicnon-healing wounds are associated with a number of biological tissuechanges including inflammation, proliferation, remodeling of connectivetissues and, a common major concern, bacterial infection. A proportionof wound infections are not clinically apparent and contribute to thegrowing economic burden associated with wound care, especially in agingpopulations. Currently, the gold-standard wound assessment includesdirect visual inspection of the wound site under white light combinedwith indiscriminate collection of bacterial swabs and tissue biopsiesresulting in delayed, costly and often insensitive bacteriologicalresults. This may affect the timing and effectiveness of treatment.Qualitative and subjective visual assessment only provides a gross viewof the wound site, but does not provide information about underlyingbiological and molecular changes that are occurring at the tissue andcellular level. A relatively simple and complementary method thatexploits ‘biological and molecular’ information to improve the earlyidentification of such occult change is desirable in clinical woundmanagement. Early recognition of high-risk wounds may guide therapeuticintervention and provide response monitoring over time, thus greatlyreducing both morbidity and mortality due especially to chronic wounds.

Wound care and management is major clinical challenge that presents asignificant burden and challenge to health care globally [Bowler et al.,Clin Microbiol Rev. 2001, 14:244-269; Cutting et al., Journal of WoundCare. 1994, 3:198-201; Dow et al., Ostomy/Wound Management. 1999,45:23-40]. Wounds are generally classified as, wounds without tissueloss (e.g. in surgery), and wounds with tissue loss, such as burnwounds, wounds caused as a result of trauma, abrasions or as secondaryevents in chronic ailments (e.g., venous stasis, diabetic ulcers orpressure sores and iatrogenic wounds such as skin graft donor sites anddermabrasions, pilonidal sinuses, non-healing surgical wounds andchronic cavity wounds). Wounds are also classified by the layersinvolved, superficial wounds involve only the epidermis, partialthickness wounds involve only epidermis and dermis, and full thicknesswounds involve the subcutaneous fat or deeper tissue. Althoughrestoration of tissue continuity after injury is a natural phenomenon,infection, quality of healing, speed of healing, fluid loss and othercomplications that enhance the healing time represents a major clinicalchallenge. The majority of wounds heal without any complication.However, chronic non-healing wounds involving progressively more tissueloss result in a large challenge for wound-care practitioners andresearchers. Unlike surgical incisions where there is relatively littletissue loss and wounds generally heal without significant complications,chronic wounds disrupt the normal process of healing which is often notsufficient in itself to effect repair. Delayed healing is generally aresult of compromised wound physiology [Winter (1962) Nature.193:293-294] and typically occurs with venous stasis and diabeticulcers, or prolonged local pressure as in immuno-suppressed andimmobilized elderly individuals. These chronic conditions increase thecost of care and reduce the patient's quality of life. As these groupsare growing in number, the need for advanced wound care products willincrease.

Conventional clinical assessment methods of acute and chronic woundscontinue to be suboptimal. They are usually based on a complete patienthistory, qualitative and subjective clinical assessment with simplevisual appraisal using ambient white light and the ‘naked eye’, and cansometimes involve the use of color photography to capture the generalappearance of a wound under white light illumination [Perednia (1991) JAm Acad Dermatol. 25: 89-108]. Regular re-assessment of progress towardhealing and appropriate modification of the intervention is alsonecessary. Wound assessment terminology is non-uniform, many questionssurrounding wound assessment remain unanswered, agreement has yet to bereached on the key wound parameters to measure in clinical practice, andthe accuracy and reliability of available wound assessment techniquesvary. Visual assessment is frequently combined with swabbing and/ortissue biopsies for bacteriological culture for diagnosis. Bacterialswabs are collected at the time of wound examination and have the notedadvantage of providing identification of specific bacterial/microbialspecies [Bowler, 2001; Cutting, 1994; Dow, 1999; Dow G. In: Krasner etal. eds. Chronic Wound Care: A Clinical Source Book for HealthcareProfessionals, 3rd ed. Wayne Pa.: HMP Communications. 2001:343-356].However, often, multiple swabs and/or biopsies are collected randomlyfrom the wound site, and some swabbing techniques may in fact spread themicroorganisms around with the wound during the collection process thusaffecting patient healing time and morbidity [Dow, 1999]. This may be aproblem especially with large chronic (non-healing) wounds where thedetection yield for bacterial presence using current swabbing and biopsyprotocols is suboptimal (diagnostically insensitive), despite many swabsbeing collected. Thus, current methods for obtaining swabs or tissuebiopsies from the wound site for subsequent bacteriological culture arebased on a non-targeted or ‘blind’ swabbing or punch biopsy approach,and have not been optimized to minimize trauma to the wound or tomaximize the diagnostic yield of the bacteriology tests. In addition,obtaining swabs and biopsy samples for bacteriology can be laborious,invasive, painful, costly, and more importantly, bacteriological cultureresults often take about 2-3 days to come back from the laboratory andcan be inconclusive [Serena et al. (2008) Int J Low Extrem Wounds.7(1):32-5.; Gardner et al., (2007) WOUNDS. 19(2):31-38], thus delayingaccurate diagnosis and treatment [Dow, 1999]. Thus, bacterial swabs donot provide real-time detection of infectious status of wounds. Althoughwound swabbing appears to be straightforward, it can lead toinappropriate treatment, patient morbidity and increased hospital staysif not performed correctly [Bowler, 2001; Cutting, 1994; Dow, 1999; Dow,2001]. The lack of a non-invasive imaging method to objectively andrapidly evaluate wound repair at a biological level (which may be atgreater detail than simply appearance or morphology based), and to aidin targeting of the collection of swab and tissue biopsy samples forbacteriology is a major obstacle in clinical wound assessment andtreatment. An alternative method is highly desirable.

As wounds (chronic and acute) heal, a number of key biological changesoccur at the wound site at the tissue and cellular level [Cutting,1994]. Wound healing involves a complex and dynamic interaction ofbiological processes divided into four overlapping phases—haemostasis,inflammation, cellular proliferation, and maturation or remodeling ofconnective tissues—which affect the pathophysiology of wound healing[Physiological basis of wound healing, in Developments in wound care,PJB Publications Ltd., 5-17, 1994]. A common major complication arisingduring the wound healing process, which can range from days to months,is infection caused by bacteria and other microorganisms [Cutting, 1994;Dow, 1999]. This can result in a serious impediment to the healingprocess and lead to significant complications. All wounds containbacteria at levels ranging from contamination, through colonization,critical colonization to infection, and diagnosis of bacterial infectionis based on clinical symptoms and signs (e.g., visual and odorous cues).

The most commonly used terms for wound infection have included woundcontamination, wound colonisation, wound infection and, more recently,critical colonisation. Wound contamination refers to the presence ofbacteria within a wound without any host reaction [Ayton M. Nurs Times1985, 81(46): suppl 16-19], wound colonisation refers to the presence ofbacteria within the wound which do multiply or initiate a host reaction[Ayton, 1985], Critical colonisation refers to multiplication ofbacteria causing a delay in wound healing, usually associated with anexacerbation of pain not previously reported but still with no overthost reaction [Falanga et al., J Invest Dermatol 1994, 102(1): 125-27;Kingsley A, Nurs Stand 2001, 15(30): 50-54, 56, 58]. Wound infectionrefers to the deposition and multiplication of bacteria in tissue withan associated host reaction [Ayton, 1985]. In practice the term‘critical colonisation’ can be used to describe wounds that areconsidered to be moving from colonisation to local infection. Thechallenge within the clinical setting, however, is to ensure that thissituation is quickly recognized with confidence and for the bacterialbioburden to be reduced as soon as possible, perhaps through the use oftopical antimicrobials. Potential wound pathogens can be categorisedinto different groups, such as, bacteria, fungi, spores, protozoa andviruses depending on their structure and metabolic capabilities [Cooperet al., Wound Infection and Microbiology.: Medical Communications (UK)Ltd for Johnson & Johnson Medical, 2003]. Although viruses do notgenerally cause wound infections, bacteria can infect skin lesionsformed during the course of certain viral diseases. Such infections canoccur in several settings including in health-care settings (hospitals,clinics) and at home or chronic care facilities. The control of woundinfections is increasingly complicated, yet treatment is not alwaysguided by microbiological diagnosis. The diversity of micro-organismsand the high incidence of polymicrobic flora in most chronic and acutewounds gives credence to the value of identifying one or more bacterialpathogens from wound cultures. The early recognition of causative agentsof wound infections can assist wound care practitioners in takingappropriate measures. Furthermore, faulty collagen formation arises fromincreased bacterial burden and results in over-vascularized friableloose granulation tissue that usually leads to wound breakdown [Sapicoet al. (1986) Diagn Microbiol Infect Dis. 5:31-38].

Accurate and clinically relevant wound assessment is an importantclinical tool, but this process currently remains a substantialchallenge. Current visual assessment in clinical practice only providesa gross view of the wound site (e.g., presence of purulent material andcrusting). Current best clinical practice fails to adequately use thecritically important objective information about underlying keybiological changes that are occurring at the tissue and cellular level(e.g., contamination, colonization, infection, matrix remodeling,inflammation, bacterial/microbial infection, and necrosis) since suchindices are i) not easily available at the time of the wound examinationand ii) they are not currently integrated into the conventional woundmanagement process. Direct visual assessment of wound health statususing white light relies on detection of color andtopographical/textural changes in and around the wound, and thus may beincapable and unreliable in detecting subtle changes in tissueremodeling. More importantly, direct visual assessment of wounds oftenfails to detect the presence of bacterial infection, since bacteria areoccult under white light illumination. Infection is diagnosed clinicallywith microbiological tests used to identify organisms and theirantibiotic susceptibility. Although the physical indications ofbacterial infection can be readily observed in most wounds using whitelight (e.g., purulent exudate, crusting, swelling, erythema), this isoften significantly delayed and the patient is already at increased riskof morbidity (and other complications associated with infection) andmortality. Therefore, standard white light direct visualization fails todetect the early presence of the bacteria themselves or identify thetypes of bacteria within the wound.

Implantation and grafting of stem cells have recently become ofinterest, such as for wound care and treatment. However, it is currentlychallenging to track the proliferation of stem cells after implantationor grafting. Tracking and identifying cancer cells have also beenchallenging. It would be desirable if such cells could be monitored in aminimally-invasive or non-invasive way.

It is also useful to provide a way for detecting contamination of othertarget surfaces, including non-biological targets.

SUMMARY

A device and method for fluorescence-based monitoring is disclosed. Insome aspects, the device comprises an optical (e.g., fluorescence and/orreflectance) device for real-time, non-invasive imaging of biochemicaland/or organic substances, for example wounds. This device may becompact, portable, and/or hand-held, and may provide high-resolutionand/or high-contrast images. Such a device may be easily integrated intocurrent wound care practice. This imaging device may rapidly andconveniently provide the clinician/health care worker with valuablebiological information of a wound: including imaging of connectivetissue changes, early detection of bacterial contamination/infection.The device may also facilitate wound margin delineation, image-guidedcollection of bacterial swab/biopsy samples, imaging of exogenousmolecular biomarker-targeted and activated optical (e.g., absorption,scattering, fluorescence, reflectance) contrast agents, and may permitlongitudinal monitoring of therapeutic response for adaptiveintervention in wound management. By exploiting wireless capabilitieswith dedicated image analysis and diagnostic algorithms, the device maybe integrated seamlessly into telemedicine (e.g., E-health)infrastructure for remote-access to specialists in wound care. Such adevice may also have applications outside wound care, including earlydetection of cancers, monitoring of emerging photodynamic therapies,detection and monitoring of stem cells, and as an instrument in thedermatology and cosmetology clinics, in addition to detecting bacteriaor contamination for other applications.

In some aspects, a method of screening for contamination during foodproduction is disclosed. The method includes applying an exogenous,bacteria-specific contrast agent to a surface to be screened, whereinthe surface to be screened is one or more of a food product, afood-preparation surface, a food-handling surface, and a food-equipmentsurface. The surface on which the contrast agent has been applied isilluminated with excitation light emitted by at least one lightexcitation light source of a handheld imaging device. With at least oneoptical filter of the handheld device, optical signals responsive toillumination of the surface including the contrast agent with theexcitation light are filtered, the at least one optical filter beingconfigured to enable optical signals having a wavelength correspondingto bacterial fluorescence to pass through the filter. Bacterialfluorescence data regarding the illuminated surface is collected with animage sensor of the handheld imaging device, wherein the collectedbacterial fluorescence data comprises at least one image containing datarelating to exogenous fluorescence of bacteria responsive toillumination of the surface. And, in real-time, based on the collectedbacterial fluorescence data, the presence and/or location of bacteria onthe illuminated surface is determined.

In some aspects, a method for fluorescence-based imaging of a target todetect contamination is disclosed, the target being selected from thegroup consisting of: a food product, a food-preparation surface, afood-handling surface, a food packaging surface, an area in a foodprocessing plant, an area in a food production facility, and afood-equipment surface. The method includes labelling a selectedbiomarker at the target with at least one fluorescing contrast agent.The target is illuminated with excitation light emitted by an excitationlight source and having at least one wavelength or wavelength bandcausing at least the selected biomarker to fluoresce. Fluorescenceemissions of at least the selected biomarker are detected with an imagedetector of a handheld imaging device. And, the presence, location,and/or quantity of contamination on the illuminated target is determinedbased on the detected fluorescence emissions of at least the selectedbiomarker.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram of a device for fluorescence-basedmonitoring;

FIG. 1b shows an example of a clinical wound care facility using adevice for fluorescence-based monitoring;

FIG. 2 shows images of a hand-held embodiment of a device forfluorescence-based monitoring;

FIG. 3 shows images of live bacterial cultures captured using a devicefor fluorescence-based monitoring;

FIG. 3J shows an example of bacterial monitoring using a device forfluorescence-based monitoring;

FIG. 4 shows images of a simulated animal wound model, demonstratingnon-invasive autofluorescence detection of bacteria using a device forfluorescence-based monitoring;

FIG. 5 shows images of a skin surface of a pig meat sample,demonstrating non-invasive autofluorescence detection of collagen andvarious bacterial species using a device for fluorescence-basedmonitoring;

FIG. 6 shows images of a muscle surface of a pig meat sample,demonstrating the use of a device for fluorescence-based monitoring forautofluorescence detection of connective tissues and bacteria;

FIG. 7 shows images and spectral plots demonstrating the use of a devicefor fluorescence-based monitoring to detect fluorescence from bacteriagrowing in agar plates and on the surface a simulated wound on pig meat;

FIG. 8 shows images of bacterial cultures demonstrating of a device forfluorescence-based monitoring, with and without contrast agents;

FIG. 9 shows images demonstrating the use of a device forfluorescence-based monitoring for autofluorescence detection ofconnective tissues and various bacterial species on the skin surface ofa pig meat sample;

FIG. 10 shows images demonstrating use of a device forfluorescence-based monitoring for fluorescence contrast-enhanceddetection of bacterial infection in a pig meat sample;

FIG. 10G shows an example of use of a device for fluorescence-basedmonitoring for monitoring effectiveness of a photodynamic treatment;

FIG. 11 shows images demonstrating use of a device forfluorescence-based monitoring for imaging of blood and microvasculature;

FIG. 12 shows images demonstrating use of a device forfluorescence-based monitoring for imaging of the oral cavity and theskin surface;

FIG. 12J shows an example of the use of a device for fluorescence-basedmonitoring for imaging a skin surface;

FIG. 13 shows images demonstrating use of a device forfluorescence-based monitoring for detection of exogenous fluorescencecontrast agents in vivo;

FIG. 14 shows images demonstrating use of a device forfluorescence-based monitoring for fluorescence-image guided surgeryusing imaging contrast agents;

FIG. 15 shows images demonstrating use of a device forfluorescence-based monitoring for video recording of fluorescence-imageguided surgery;

FIG. 16 shows images demonstrating use of a device forfluorescence-based monitoring for autofluorescence-image guided surgicalresections of tissues in a mouse cardiac infarction model;

FIG. 17 shows images demonstrating use of a device forfluorescence-based monitoring for autofluorescence-image guided surgeryof a mouse brain;

FIG. 18 shows images demonstrating the use of a device forfluorescence-based monitoring in imaging cancer stem cells in a mouse;

FIG. 19 shows images demonstrating the use of a device forfluorescence-based monitoring in imaging cancer stem cells in a liverand a lung;

FIGS. 19H and 19I show examples of the use of a device forfluorescence-based monitoring for imaging tumours;

FIG. 20 shows images demonstrating the use of a device forfluorescence-based monitoring in imaging a mouse model;

FIG. 20B shows an example of the use of a device for fluorescence-basedmonitoring for imaging small animal models;

FIG. 21 illustrates the phases of wound healing with time;

FIG. 22 is a table showing examples of tissue, cellular and molecularbiomarkers known to be associated with wound healing;

FIG. 23 is a diagram comparing a healthy wound to a chronic wound;

FIG. 24 illustrates an example of monitoring of a chronic wound;

FIGS. 24B-24P show examples of the use of a device forfluorescence-based monitoring for imaging wounds and conditions inclinical patients;

FIG. 24Q shows an example of the use of a device for fluorescence-basedmonitoring for imaging bacterial response to photodynamic therapy;

FIG. 24R shows an example of the use of a device for fluorescence-basedmonitoring for imaging tissue;

FIG. 25 is a flowchart illustrating the management of a chronic woundusing a device for fluorescence-based monitoring;

FIGS. 26 and 27 show examples of the use of a device forfluorescence-based monitoring for detecting contamination in foodproducts;

FIGS. 28 and 28B-28C show examples of the use of a device forfluorescence-based monitoring for detecting surface contamination;

FIGS. 29-31 show examples of the use of a device for fluorescence-basedmonitoring for forensic applications;

FIG. 32 shows an example of the use of a device for fluorescence-basedmonitoring for cataloguing animals;

FIG. 33 shows an example of a kit including a device forfluorescence-based monitoring; and

FIG. 34 shows an example of the use of a device for fluorescence-basedmonitoring for imaging cosmetic or dermatological substances.

DETAILED DESCRIPTION

Wound progression is currently monitored manually. The National PressureUlcer Advisory Panel (NPUAP) developed the Pressure Ulcer Scale forHealing (PUSH) tool that outlines a five-step method of characterizingpressure ulcers. This tool uses three parameters to determine aquantitative score that is then used to monitor the pressure ulcer overtime. The qualitative parameters include wound dimensions, tissue type,and the amount of exudate or discharge, and thermal readings presentafter the dressing is removed. A wound can be further characterized byits odor and color. Such an assessment of wounds currently does notinclude critical biological and molecular information about the wound.Therefore, all descriptions of wounds are somewhat subjective and notedby hand by either the attending physician or the nurse.

What is desirable is a robust, cost-effective non-invasive and rapidimaging-based method or device for objectively assessing wounds forchanges at the biological, biochemical and cellular levels and forrapidly, sensitively and non-invasively detecting the earliest presenceof bacteria/microorganisms within wounds. Such a method or device fordetection of critical biological tissue changes in wounds may serve anadjunctive role with conventional clinical wound management methods inorder to guide key clinico-pathological decisions in patient care. Sucha device may be compact, portable and capable of real-time non-invasiveand/or non-contact interrogation of wounds in a safe and convenientmanner, which may allow it to fit seamlessly into routine woundmanagement practice and user friendly to the clinician, nurse and woundspecialist. This may also include use of this device in the home-careenvironment (including self-use by a patient), as well as in militarybattlefield environments. In addition, such an image-based device mayprovide an ability to monitor wound treatment response and healing inreal-time by incorporating valuable ‘biologically-informed’image-guidance into the clinical wound assessment process. This mayultimately lead to potential new diagnosis, treatment planning,treatment response monitoring and thus ‘adaptive’ interventionstrategies which may permit enhancement of wound-healing response at theindividual patient level. Precise identification of the systemic, local,and molecular factors underlying the wound healing problem in individualpatients may allow better tailored treatment.

A number of imaging technologies have become available that offer thepotential to satisfy the requirements for improved clinical diagnosisand treatment of disease. Of these, fluorescence imaging appears to bepromising for improving clinical wound assessment and management. Whenexcited by short wavelength light (e.g., ultraviolet or short visiblewavelengths), most endogenous biological components of tissues (e.g.,connective tissues such collagen and elastins, metabolic co-enzymes,proteins, etc.) produce fluorescence of a longer wavelength, in theultraviolet, visible, near-infrared and infrared wavelength ranges[DaCosta et al., Photochem Photobiol. 2003 October, 78(4):384-92]. Themost clinically mature of emerging optically-based imaging technologies,tissue autofluorescence imaging has been used to improve the endoscopicdetection of early cancers and other diseases in the gastrointestinaltract [Dacosta (2002) J Gastroenterol Hepatol. Suppl:S85-104], the oralcavity [Poh et al., Head Neck. 2007 January, 29(1):71-6], and lungs[Hanibuchi et al., (2007) J Med Invest. 54:261-6] and bladder[D′Hallewin et al. (2002) Eur Urol. 42(5):417-25] in aminimally-invasive manner.

Tissue autofluorescence imaging provides a unique means of obtainingbiologically relevant information of normal and diseased tissues inreal-time, thus allowing differentiation between normal and diseasedtissue states [DaCosta, 2003; DaCosta et al. J Clin Pathol. 2005,58(7):766-74]. This is based, in part, on the inherently differentlight-tissue interactions (e.g., abosption and scattering of light) thatoccur at the bulk tissue and cellular levels, changes in the tissuemorphology and alterations in the blood content of the tissues. Intissues, blood is a major light absorbing tissue component (i.e., achromophore). This type of technology is suited for imaging disease inhollow organs (e.g., GI tract, oral cavity, lungs, bladder) or exposedtissue surfaces (e.g., skin) Despite this indication, current endoscopicfluorescence imaging systems are large, involve complex diagnosticalgorithms and expensive, and to date, such instruments are mainly foundin large clinical centers and very few systems are commerciallyavailable. Currently, no such optical or fluorescence-based imagingdevice exists for wound imaging. However, since wounds are readilyaccessible, an autofluorescence imaging device may be useful for rapid,non-invasive and non-contact real-time imaging of wounds, to detect andexploit the rich biological information of the wound to overcome currentlimitations and improve clinical care and management.

A method and device for fluorescence-based imaging and monitoring isdisclosed. One embodiment of the device is a portable optical digitalimaging device. The device may utilize a combination of white light,tissue fluorescence and reflectance imaging, and may provide real-timewound imaging, assessment, recording/documenting, monitoring and/or caremanagement. The device may be hand-held, compact and/or light-weight.This device and method may be suitable for monitoring of wounds inhumans and animals.

Other uses for the device may include:

-   -   Clinically- and research-based imaging of small and large (e.g.,        veterinary) animals.    -   Detection and monitoring of contamination (e.g., bacterial        contamination) in food/animal product preparation in the meat,        poultry, dairy, fish, agricultural industries.    -   Detection of ‘surface contamination’ (e.g., bacterial or        biological contamination) in public (e.g., health care) and        private settings.    -   Multi-spectral imaging and detection of cancers in human and/or        veterinary patients.    -   As a research tool for multi-spectral imaging and monitoring of        cancers in experimental animal models of human diseases (e.g.,        wound and cancers).    -   Forensic detection, for example of latent finger prints and        biological fluids on non-biological surfaces.    -   Imaging and monitoring of dental plaques, carries and cancers in        the oral cavity.    -   Imaging and monitoring device in clinical microbiology        laboratories.    -   Testing anti-bacterial (e.g., antibiotic), disinfectant agents.

The device may generally comprise: i) one or moreexcitation/illumination light sources and ii) a detector device (e.g., adigital imaging detector device), which may be combined with one or moreoptical emission filters, or spectral filtering mechanisms, and whichmay have a view/control screen (e.g., a touch-sensitive screen), imagecapture and zoom controls. The device may also have: iii) a wired and/orwireless data transfer port/module, iv) an electrical power source andpower/control switches, and/or v) an enclosure, which may be compactand/or light weight, and which may have a mechanism for attachment ofthe detector device and/or a handle grip. The excitation/illuminationlight sources may be LED arrays emitting light at about 405 nm (e.g.,+/−5 nm), and may be coupled with additional band-pass filters centeredat about 405 nm to remove/minimize the side spectral bands of light fromthe LED array output so as not to cause light leakage into the imagingdetector with its own optical filters. The digital imaging detectordevice may be a digital camera, for example having at least an ISO800sensitivity, but more preferably an ISO3200 sensitivity, and may becombined with one or more optical emission filters, or other equallyeffective (e.g., miniaturized) mechanized spectral filtering mechanisms(e.g., acousto-optical tunable filter or liquid crystal tunable filter).The digital imaging detector device may have a touch-sensitive viewingand/or control screen, image capture and zoom controls. The enclosuremay be an outer hard plastic or polymer shell, enclosing the digitalimaging detector device, with buttons such that all necessary devicecontrols may be accessed easily and manipulated by the user. Miniatureheat sinks or small mechanical fans, or other heat dissipating devicesmay be imbedded in the device to allow excess heat to be removed fromthe excitation light sources if required. The complete device, includingall its embedded accessories and attachments, may be powered usingstandard AC/DC power and/or by rechargeable battery pack. The completedevice may also be attached or mounted to an external mechanicalapparatus (e.g., tripod, or movable stand with pivoting arm) allowingmobility of the device within a clinical room with hands-free operationof the device. Alternatively, the device may be provided with a mobileframe such that it is portable. The device may be cleaned using moistgauze wet with water, while the handle may be cleansed with moist gauzewet with alcohol. The device may include software allowing a user tocontrol the device, including control of imaging parameters,visualization of images, storage of image data and user information,transfer of images and/or associated data, and/or relevant imageanalysis (e.g., diagnostic algorithms)

A schematic diagram of an example of the device is shown in FIG. 1. Thedevice is shown positioned to image a target object 10 or targetsurface. In the example shown, the device has a digital imageacquisition device 1, such as digital camera, video recorder, camcorder,cellular telephone with built-in digital camera, ‘Smart’ phone with adigital camera, personal digital assistant (PDA), laptop/PC with adigital camera, or a webcam. The digital image acquisition device 1 hasa lens 2, which may be aligned to point at the target object 10 and maydetect the optical signal that emanates from the object 10 or surface.The device has an optical filter holder 3 which may accommodate one ormore optical filters 4. Each optical filter 4 may have differentdiscrete spectral bandwidths and may be band-pass filters. These opticalfilters 4 may be selected and moved in from of the digital camera lensto selectively detect specific optical signals based on the wavelengthof light. The device may include light sources 5 that produce excitationlight to illuminate the object 10 in order to elicit an optical signal(e.g., fluorescence) to be imaged with, for example, blue light (e.g.,400-450 nm), or any other combination of single or multiple wavelengths(e.g., wavelengths in the ultraviolet/visible/near infrared/infraredranges). The light source 5 may comprise a LED array, laser diode and/orfiltered lights arranged in a variety of geometries. The device mayinclude a method or apparatus 6 (e.g., a heatsink or a cooling fan) todissipate heat and cool the illumination light sources 5. The device mayinclude a method or apparatus 7 (e.g., an optical band-pass filter) toremove any undesirable wavelengths of light from the light sources 5used to illuminate the object 10 being imaged. The device may include amethod or apparatus 8 to use an optical means (e.g., use of compactminiature laser diodes that emit a collimated light beam) to measure anddetermine the distance between the imaging device and the object 10. Forexample, the device may use two light sources, such as two laser diodes,as part of a triangulation apparatus to maintain a constant distancebetween the device and the object 10. Other light sources may bepossible. The device may also use ultrasound, or a physical measure,such as a ruler, to determine a constant distance to maintain. Thedevice may also include a method or apparatus 9 (e.g., a pivot) topermit the manipulation and orientation of the excitation light sources5, 8 so as to manoeuvre these sources 5,8 to change the illuminationangle of the light striking the object 10 for varying distances.

The target object 10 may be marked with a mark 11 to allow for multipleimages to be taken of the object and then being co-registered foranalysis. The mark 11 may involve, for example, the use of exogenousfluorescence dyes of different colours which may produce multipledistinct optical signals when illuminated by the light sources 5 and bedetectable within the image of the object 10 and thus may permitorientation of multiple images (e.g., taken over time) of the sameregion of interest by co-registering the different colours and thedistances between them. The digital image acquisition device 1 mayinclude one or more of: an interface 12 for a head-mounted display; aninterface 13 for an external printer; an interface 14 for a tabletcomputer, laptop computer, desk top computer or other computer device;an interface 15 for the device to permit wired or wireless transfer ofimaging data to a remote site or another device; an interface 16 for aglobal positioning system (GPS) device; an interface 17 for a deviceallowing the use of extra memory; and an interface 18 for a microphone.

The device may include a power supply 19 such as an AC/DC power supply,a compact battery bank, or a rechargeable battery pack. Alternatively,the device may be adapted for connecting to an external power supply.The device may have a housing 20 that houses all the components in oneentity. The housing 20 may be equipped with a means of securing anydigital imaging device within it. The housing 20 may be designed to behand-held, compact, and/or portable. The housing 20 may be one or moreenclosures.

Referring still to FIG. 1, b) shows an example of the device in atypical wound care facility. a) shows a typical clinical wound carefacility, showing the examination chair and accessory table. b-c) Anexample of the device is shown in its hard-case container. The devicemay be integrated into the routine wound care practice allowingreal-time imaging of the patient. d) An example of the device (arrow) isshown placed on the “wound care cart” to illustrate the size of thedevice. e) The device may be used to image under white lightillumination, while f) shows the device being used to take fluorescenceimages of a wound under dimmed room lights. g) The device may be used intelemedicine/telehealth infrastructures, for example fluorescence imagesof a patient's wounds may be sent by email to a wound care specialistvia a wireless communication device, such as a Smartphone at anotherhospital using a wireless/WiFi internet connection. Using this device,high-resolution fluorescence images may be sent as email attachments towound care specialists from remote wound care sites for immediateconsultation with clinical experts, microbiologists, etc. at specializedclinical wound care and management centers.

EXAMPLES

An example of a device for fluorescence-based monitoring is describedbelow. All examples are provided for the purpose of illustration onlyand are not intended to be limiting. Parameters such as wavelengths,dimensions, and incubation time described in the examples may beapproximate and are provided as examples only.

In this example, the devices uses two violet/blue light (e.g., 405mm+/−10 nm emission, narrow emission spectrum) LED arrays (Opto DiodeCorporation, Newbury Park, Calif.), each situated on either side of theimaging detector assembly as the excitation or illumination lightsources. These arrays have an output power of approximately 1 Watt each,emanating from a 2.5×2.5 cm², with a 70-degree illuminating beam angle.The LED arrays may be used to illuminate the tissue surface from adistance of about 10 cm, which means that the total optical powerdensity on the skin surface is about 0.08 W/cm². At such low powers,there is no known potential harm to either the target wound or skinsurface, or the eyes from the excitation light. However, it may beinadvisable to point the light directly at any individual's eyes duringimaging procedures. It should also be noted that 405 nm light does notpose a risk to health according to international standards formulated bythe International Electrotechnical Commission (IEC) , as furtherdetailed on the website:

http://www.iec.ch/online_news/etech/arch_2006/etech_0906/focus.htm

The one or more light sources may be articulated (e.g., manually) tovary the illumination angle and spot size on the imaged surface, forexample by using a built in pivot, and are powered for example throughan electrical connection to a wall outlet and/or a separate portablerechargeable battery pack. Excitation/illumination light may be producedby sources including, but not limited to, individual or multiplelight-emitting diodes (LEDs) in any arrangement including in ring orarray formats, wavelength-filtered light bulbs, or lasers. Selectedsingle and multiple excitation/illumination light sources with specificwavelength characteristics in the ultraviolet (UV), visible (VIS),far-red, near infrared (NIR) and infrared (IR) ranges may also be used,and may be composed of a LED array, organic LED, laser diode, orfiltered lights arranged in a variety of geometries.Excitation/illumination light sources may be ‘tuned’ to allow the lightintensity emanating from the device to be adjusted while imaging. Thelight intensity may be variable. The LED arrays may be attached toindividual cooling fans or heat sinks to dissipate heat produced duringtheir operation. The LED arrays may emit narrow 405 nm light, which maybe spectrally filtered using a commercially available band-pass filter(Chroma Technology Corp, Rockingham, Vt., USA) to reduce potential‘leakage’ of emitted light into the detector optics. When the device isheld above a tissue surface (e.g., a wound) to be imaged, theilluminating light sources may shine a narrow-bandwidth orbroad-bandwidth violet/blue wavelength or other wavelength or wavelengthband of light onto the tissue/wound surface thereby producing a flat andhomogeneous field within the region-of-interest. The light may alsoilluminate or excite the tissue down to a certain shallow depth. Thisexcitation/illumination light interacts with the normal and diseasedtissues and may cause an optical signal (e.g., absorption, fluorescenceand/or reflectance) to be generated within the tissue.

By changing the excitation and emission wavelengths accordingly, theimaging device may interrogate tissue components (e.g., connectivetissues and bacteria in a wound) at the surface and at certain depthswithin the tissue (e.g., a wound). For example, by changing fromviolet/blue (˜400-500 nm) to green (˜500-540 nm) wavelength light,excitation of deeper tissue/bacterial fluorescent sources may beachieved, for example in a wound. Similarly, by detecting longerwavelengths, fluorescence emission from tissue and/or bacterial sourcesdeeper in the tissue may be detected at the tissue surface. For woundassessment, the ability to interrogate surface and/or sub-surfacefluorescence may be useful, for example in detection and potentialidentification of bacterial contamination, colonization, criticalcolonization and/or infection, which may occur at the surface and oftenat depth within a wound (e.g., in chronic non-healing wounds). In oneexample, Referring to FIG. 6, c) shows the detection of bacteria belowthe skin surface (i.e., at depth) after wound cleaning. This use of thedevice for detecting bacteria at the surface and at depth within a woundand surrounding tissue may be assessed in the context of other clinicalsigns and symptoms used conventionally in wound care centers.

Example embodiments of the device are shown in FIG. 2. The device may beused with any standard compact digital imaging device (e.g., acharge-coupled device (CCD) or complementary metal-oxide-semiconductor(CMOS) sensors) as the image acquisition device. The example deviceshown in a) has an external electrical power source, the two LED arraysfor illuminating the object/surface to be imaged, and a commerciallyavailable digital camera securely fixed to light-weight metal frameequipped with a convenient handle for imaging. A multi-band filter isheld in front of the digital camera to allow wavelength filtering of thedetected optical signal emanating from the object/surface being imaged.The camera's video/USB output cables allow transfer of imaging data to acomputer for storage and subsequent analysis. This example uses acommercially-available 8.1-megapixel Sony digital camera (Sony CybershotDSC-T200 Digital Camera, Sony Corporation, North America). This cameramay be suitable because of i) its slim vertical design which may beeasily integrated into the enclosure frame, ii) its large 3.5-inchwidescreen touch-panel LCD for ease of control, iii) its Carl Zeiss 5×optical zoom lens, and iv) its use in low light (e.g., ISO 3200). Thedevice may have a built-in flash which allows for standard white lightimaging (e.g., high-definition still or video with sound recordingoutput). Camera interface ports may support both wired (e.g., USB) orwireless (e.g., Bluetooth, WiFi, and similar modalities) data transferor 3^(rd) party add-on modules to a variety of external devices, suchas: a head-mounted display, an external printer, a tablet computer,laptop computer, personal desk top computer, a wireless device to permittransfer of imaging data to a remote site/other device, a globalpositioning system (GPS) device, a device allowing the use of extramemory, and a microphone. The digital camera is powered by rechargeablebatteries, or AC/DC powered supply. The digital imaging device mayinclude, but is not limited to, digital cameras, webcams, digital SLRcameras, camcorders/video recorders, cellular telephones with embeddeddigital cameras, Smartphones™, personal digital assistants (PDAs), andlaptop computers/tablet PCs, or personal desk-top computers, all ofwhich contain/or are connected to a digital imaging detector/sensor.

This light signal produced by the excitation/illumination light sourcesmay be detected by the imaging device using optical filter(s) (e.g.,those available from Chroma Technology Corp, Rockingham, Vt., USA) thatreject the excitation light but allow selected wavelengths of emittedlight from the tissue to be detected, thus forming an image on thedisplay. There is an optical filter holder attached to the enclosureframe in from of the digital camera lens which may accommodate one ormore optical filters with different discrete spectral bandwidths, asshown in b) and c) of FIG. 2. b) shows the device with the LED arraysturned on to emit bright violet/blue light, with a single emissionfilter in place. c) shows the device using a multiple-optical filterholder used to select the appropriate filter for desiredwavelength-specific imaging. d) shows the device being held in one handwhile imaging the skin surface of a foot.

These band-pass filters may be selected and aligned in front of thedigital camera lens to selectively detect specific optical signals fromthe tissue/wound surface based on the wavelength of light desired.Spectral filtering of the detected optical signal (e.g., absorption,fluorescence, reflectance) may also be achieved, for example, using aliquid crystal tunable filter (LCTF), or an acousto-optic tunable filter(AOTF) which is a solid-state electronically tunable spectral band-passfilter. Spectral filtering may also involve the use of continuousvariable filters, and/or manual band-pass optical filters. These devicesmay be placed in front of the imaging detector to produce multispectral,hyperspectral, and/or wavelength-selective imaging of tissues.

The device may be modified by using optical or variably orientedpolarization filters (e.g., linear or circular combined with the use ofoptical wave plates) attached in a reasonable manner to theexcitation/illumination light sources and the imaging detector device.In this way, the device may be used to image the tissue surface withpolarized light illumination and non-polarized light detection or viceversa, or polarized light illumination and polarized light detection,with either white light reflectance and/or fluorescence imaging. Thismay permit imaging of wounds with minimized specular reflections (e.g.,glare from white light imaging), as well as enable imaging offluorescence polarization and/or anisotropy-dependent changes inconnective tissues (e.g., collagens and elastin) within the wound andsurrounding normal tissues. This may yield useful information about thespatial orientation and organization of connective tissue fibersassociated with wound remodeling during healing [Yasui et al., (2004)Appl. Opt. 43: 2861-2867].

All components of the imaging device may be integrated into a singlestructure, such as an ergonomically designed enclosed structure with ahandle, allowing it to be comfortably held with one or both hands. Thedevice may also be provided without any handle. The device may be lightweight, portable, and may enable real-time digital imaging (e.g., stilland/or video) of any target surface (for example, the skin and/or oralcavity, which is also accessible) using white light, fluorescence and/orreflectance imaging modes. The device may be scanned across the bodysurface for imaging by holding it at variable distances from thesurface, and may be used in a lit environment/room to image white lightreflectance/fluorescence. The device may be used in a dim or darkenvironment/room to optimize the tissue fluorescence signals, andminimize background signals from room lights. The device may be used fordirect (e.g., with the unaided eye) or indirect (e.g., via the viewingscreen of the digital imaging device) visualization of wounds andsurrounding normal tissues.

The device may also be embodied as not being hand-held or portable, forexample as being attached to a mounting mechanism (e.g., a tripod orstand) for use as a relatively stationary optical imaging device forwhite light, fluorescence and reflectance imaging of objects, materials,and surfaces (e.g., a body). This may allow the device to be used on adesk or table or for ‘assembly line’ imaging of objects, materials andsurfaces. In some embodiments, the mounting mechanism may be mobile.

Other features of this device may include the capability of digitalimage and video recording, possibly with audio, methods fordocumentation (e.g., with image storage and analysis software), andwired or wireless data transmission for remote telemedicine/E-healthneeds. For example, e) and f) of FIG. 2 show an embodiment of the devicewhere the image acquisition device is a mobile communication device suchas a cellular telephone. The cellular telephone used in this example isa Samsung Model A-900, which is equipped with a 1.3 megapixel digitalcamera. The telephone is fitted into the holding frame for convenientimaging. e) shows the use of the device to image a piece of paper withfluorescent ink showing the word “Wound”. f) shows imaging offluorescent ink stained fingers, and detection of the common skinbacteria P. acnes. The images from the cellular telephone may be sentwirelessly to another cellular telephone, or wirelessly (e.g., viaBluetooth connectivity) to a personal computer for image storage andanalysis. This demonstrates the capability of the device to performreal-time hand-held fluorescence imaging and wireless transmission to aremote site/person as part of a telemedicine/E-health wound careinfrastructure.

In order to demonstrate the capabilities of the imaging device in woundcare and other relevant applications, a number of feasibilityexperiments were conducted using the particular example described. Itshould be noted that during all fluorescence imaging experiments, theSony camera (Sony Cybershot DSC-T200 Digital Camera, Sony Corporation,North America) settings were set so that images were captured without aflash, and with the ‘Macro’ imaging mode set. Images were captured at 8megapixels. The flash was used to capture white light reflectanceimages. All images were stored on the xD memory card for subsequenttransfer to a personal computer for long-term storage and imageanalysis.

All white light reflectance and fluorescence images/movies captured withthe device were imported into Adobe Photoshop for image analysis.However, image analysis software was designed using MatLab™ (Mathworks)to allow a variety of image-based spectral algorithms (e.g.,red-to-green fluorescence ratios, etc.) to be used to extract pertinentimage data (e.g., spatial and spectral data) for quantitativedetection/diagnostic value. Image post-processing also includedmathematical manipulation of the images.

Imaging of Bacteriological Samples

The imaging device may be useful for imaging and/or monitoring inclinical microbiology laboratories. The device may be used forquantitative imaging of bacterial colonies and quantifying colony growthin common microbiology assays. Fluorescence imaging of bacterialcolonies may be used to determine growth kinetics. Software may be usedto provide automatic counting of bacterial colonies.

To demonstration the utility of the device in a bacteriology/culturelaboratory, live bacterial cultures were grown on sheep's blood agarplates. Bacterial species included streptococcus pyogenes, serratiamarcescens, staphylococcus aureus, staphylococcus epidermidis,escherichia coli, and pseudomonas aeruginosa (American Type CultureCollection, ATCC). These were grown and maintained under standardincubation conditions at 37° C. and used for experimentation when during‘exponential growth phase’. Once colonies were detected in the plates(˜24 h after inoculation), the device was used to image agar platescontaining individual bacterial species in a darkened room. Usingviolet/blue (about 405 nm) excitation light, the device was used toimage both combined green and red autofluorescence (about 490-550 nm andabout 610-640 nm emission) and only red autofluorescence (about 635+/−10nm, the peak emission wavelength for fluorescent endogenous porphyrins)of each agar plate. Fluorescence images were taken of each bacterialspecies over time for comparison and to monitor colony growth.

Reference is now made to FIG. 3. a) shows the device being used to imagelive bacterial cultures growing on sheep's blood agar plates to detectbacterial autofluorescence. b) shows the image of autofluorescenceemitted by pseudomonas aeruginosa. The device may also be used todetect, quantify and/or monitor bacterial colony growth over time usingfluorescence, as demonstrated in c) with fluorescence imaging of thegrowth of autofluorescent staphylococcus aureus on an agar plate 24hours after inoculation. Note the presence of distinct single bacterialcolonies in the lower image. Using violet/blue (e.g., 405 nm) excitationlight, the device was used to detect both combined green and red (e.g.,490-550 nm+610-640 nm) and only red (e.g., 635+/−10 nm, the peakemission wavelength for fluorescent endogenous porphyrins) emissionautofluorescence from several live bacterial species includingstreptococcus pyogenes, shown in d); serratia marcescens, shown in e);staphylococcus aureus, shown in f); staphylococcus epidermidis, shown ing); escherichia coli, shown in h); and pseudomonas aeruginosa, shown ini). Note that the autofluorescence images obtained by the device of thebacterial colonies may provide useful image contrast for simplelongitudinal quantitative measurements of bacterial colonization andgrowth kinetics, as well as a means of potentially monitoring responseto therapeutic intervention, with antibiotics, photodynamic therapy(PDT), low level light therapy, hyperbaric oxygen therapy (HOT), oradvanced wound care products, as examples.

High spatial resolution of the camera detector combined with significantbacterial autofluorescence signal-to-noise imaging with the deviceallowed detection of very small (e.g., <1 mm diameter) colonies. Thedevice provided a portable and sensitive means of imaging individualbacterial colonies growing in standard agar plates. This provided ameans to quantify and monitor bacterial colony growth kinetics, as seenin c), as well as potentially monitoring response to therapeuticintervention, with antibiotics or photodynamic therapy (PDT) asexamples, over time using fluorescence. Therefore, the device may serveas a useful tool in the microbiology laboratory.

FIG. 3J shows an example of the use of the imaging device in a) standardbacteriology laboratory practice. b) Here, fluorescence imaging of aPetri dish containing Staphylococcus aureus combined with customproprietary image analysis software allows bacterial colonies to becounted rapidly, and here the fluorescence image of the culture dishshows ˜182 (+/−3) colonies (bright bluish-green spots) growing on agarat 37° C. (about 405 nm excitation, about 500-550 nm emission (green),about >600 nm emission (red)).

In addition to providing detecting of bacterial strains, the device maybe used for differentiating the presence and/or location of differentbacterial strains (e.g., Staphylococcus aureus or Pseudomonasaeruginosa), for example in wounds and surrounding tissues. This may bebased on the different autofluorescence emission signatures of differentbacterial strains, including those within the 490-550 nm and 610-640 nmemission wavelength bands when excited by violet/blue light, such aslight around 405 nm. Other combinations of wavelengths may be used todistinguish between other species on the images. This information may beused to select appropriate treatment, such as choice of antibiotic.

Such imaging of bacteriology samples may be applicable to monitoring ofwound care.

Use in Monitoring of Wound Healing

The device may be scanned above any wound (e.g., on the body surface)such that the excitation light may illuminate the wound area. The woundmay then be inspected using the device such that the operator may viewthe wound in real-time, for example, via a viewer on the imaging deviceor via an external display device (e.g., heads-up display, a televisiondisplay, a computer monitor, LCD projector or a head-mounted display).It may also be possible to transmit the images obtained from the devicein real-time (e.g., via wireless communication) to a remote viewingsite, for example for telemedicine purposes, or send the images directlyto a printer or a computer memory storage. Imaging may be performedwithin the routine clinical assessment of patient with a wound.

Prior to imaging, fiduciary markers (e.g., using an indeliblefluorescent ink pen) may be placed on the surface of the skin near thewound edges or perimeter. For example, four spots, each of a differentfluorescent ink color from separate indelible fluorescent ink pens,which may be provided as a kit to the clinical operator, may be placednear the wound margin or boundary on the normal skin surface. Thesecolors may be imaged by the device using the excitation light and amultispectral band filter that matches the emission wavelength of thefour ink spots. Image analysis may then be performed, by co-registeringthe fiduciary markers for inter-image alignment. Thus, the user may nothave to align the imaging device between different imaging sessions.This technique may facilitate longitudinal (i.e., over time) imaging ofwounds, and the clinical operator may therefore be able to image a woundover time without need for aligning the imaging device during everyimage acquisition.

In addition, to aid in intensity calibration of the fluorescence images,a disposable simple fluorescent standard ‘strip’ may be placed into thefield of view during wound imaging (e.g., by using a mild adhesive thatsticks the strip to the skin temporarily). The strip may be impregnatedwith one or several different fluorescent dyes of varying concentrationswhich may produce pre-determined and calibrated fluorescence intensitieswhen illuminated by the excitation light source, which may have single(e.g., 405 nm) or multiple fluorescence emission wavelengths orwavelength bands for image intensity calibration. The disposable stripmay also have the four spots as described above (e.g., each of differentdiameters or sizes and each of a different fluorescent ink color with aunique black dot placed next to it) from separate indelible fluorescentink pens. With the strip placed near the wound margin or boundary on thenormal skin surface, the device may be used to take white light andfluorescence images. The strip may offer a convenient way to takemultiple images over time of a given wound and then align the imagesusing image analysis. Also, the fluorescent ‘intensity calibration’strip may also contain an added linear measuring apparatus, such as aruler of fixed length to aid in spatial distance measurements of thewounds. Such a strip may be an example of a calibration target which maybe used with the device to aid in calibration or measuring of imageparameters (e.g., wound size, fluorescence intensity, etc.), and othersimilar calibration target may be used.

It may be desirable to increase the consistency of imaging results andto reproduce the distance between the device and the wound surface,since tissue fluorescence intensity may vary slightly if the distancechanges during multiple imaging sessions. Therefore, in an embodiment,the device may have two light sources, such as low power laser beams,which may be used to triangulate individual beams onto the surface ofthe skin in order to determine a fixed or variable distance between thedevice and the wound surface. This may be done using a simply geometricarrangement between the laser light sources, and may permit the clinicaloperator to easily visualize the laser targeting spots on the skinsurface and adjust the distance of the device from the wound duringmultiple imaging sessions. Other methods of maintaining a constantdistance may include the use of ultrasound, or the use of a physicalmeasure, such as a ruler.

Use in White Light Imaging

The device may be used to take white light images of the total woundwith normal surrounding normal tissues using a measuring apparatus(e.g., a ruler) placed within the imaging field of view. This may allowvisual assessment of the wound and calculation/determination ofquantitative parameters such as the wound area, circumference, diameter,and topographic profile. Wound healing may be assessed by planimetricmeasurements of the wound area at multiple time points (e.g., atclinical visits) until wound healing. The time course of wound healingmay be compared to the expected healing time calculated by the multipletime point measurements of wound radius reduction using the equationR=√A/π (R, radius; A, planimetric wound area; π, constant 3.14). Thisquantitative information about the wound may be used to track andmonitor changes in the wound appearance over time, in order to evaluateand determine the degree of wound healing caused by natural means or byany therapeutic intervention. This data may be stored electronically inthe health record of the patient for future reference. White lightimaging may be performed during the initial clinical assessment of thepatient by the operator.

Use in Autofluorescence Imaging

The device may be designed to detect all or a majority of tissueautofluorescence (AF). For example, using a multi-spectral band filter,the device may image tissue autofluorescence emanating from thefollowing tissue biomolecules, as well as blood-associated opticalabsorption, for example under 405 nm excitation: collagen (Types I, II,III, IV, V and others) which appear green, elastin which appearsgreenish-yellow-orange, reduced nicotinamide adenine dinucleotide(NADH), flavin adenine dinucleotide (FAD), which emit a blue-greenautofluorescence signal, and bacteria/microorganisms, most of whichappear to have a broad (e.g., green and red) autofluorescence emission.

Image analysis may include calculating a ratio of red-to-green AF in theimage. Intensity calculations may be obtained from regions of interestwithin the wound images. Pseudo-coloured images may be mapped onto thewhite light images of the wound.

Examples in Wound Healing

Reference is now made to FIG. 4. The device was tested in model ofwounds contaminated with bacteria. For this, pig meat, with skin, waspurchased from a butcher. To simulate wounds, a scalpel was used to makeincisions, ranging in size from 1.5 cm² to 4 cm² in the skin, and deepenough to see the muscle layer. The device was used to image some meatsamples without addition of bacteria to the simulated wounds. For this,the meat sample was left at room temperature for 24 h in order forbacteria on the meat to grow, and then imaging was performed with thedevice using both white light reflectance and autofluorescence, forcomparison.

To test the ability of the device to detect connective tissues andseveral common bacteria present in typical wounds, a sample of pig meatwith simulated wounds was prepared by applying six bacterial species toeach of six small 1.5 cm² wound incision sites on the skin surface:streptococcus pyogenes, serratia marcescens, staphylococcus aureus,staphylococcus epidermidis, escherichia coli, and pseudomonasaeruginosa. An additional small incision was made in the meat skin,where no bacteria were added, to serve as a control. However, it wasexpected that bacteria from the other six incisions sites would perhapscontaminate this site in time. The device was used to image thebacteria-laden meat sample using white light reflectance and violet/bluelight-induced tissue autofluorescence emission, using both a dualemission band (450-505 nm and 590-650 nm) emission filter and a singleband (635+/−10 nm) emission filter, on the left and a single band filterover the course of three days, at 24 h time intervals, during which themeat sample was maintained at 37° C. Imaging was also performed on thestyrofoam container on which the meat sample was stored during the threedays.

FIG. 4 shows the results of the device being used for non-invasiveautofluorescence detection of bacteria in a simulated animal woundmodel. Under standard white light imaging, bacteria were occult withinthe wound site, as shown in a) and magnified in b). However, underviolet/blue excitation light, the device was capable of allowingidentification of the presence of bacteria within the wound site basedon the dramatic increase in red fluorescence from bacterial porphyrinsagainst a bright green fluorescence background from connective tissue(e.g., collagen and elastins) as seen in c) and magnified in d).Comparison of b) and d) shows a dramatic increase in red fluorescencefrom bacterial porphyrins against a bright green fluorescence backgroundfrom connective tissue (e.g., collagen and elastins). It was noted thatwith autofluorescence, bacterial colonies were also detected on the skinsurface based on their green fluorescence emission causing individualcolonies to appear as punctuate green spots on the skin. These were notseen under white light examination. Fluorescence imaging of connectivetissues aided in determining the wound margins as seen in e) and f), andsome areas of the skin (marked ‘*’ in c) appeared more red fluorescentthan other areas, potentially indicating subcutaneous infection ofporphyrin-producing bacteria. e) and f) also show the device detectingred fluorescent bacteria within the surgical wound, which are occultunder white light imaging.

The device mapped biodistribution of bacteria within the wound site andon the surrounding skin and thus may aid in targeting specific tissueareas requiring swabbing or biopsy for microbiological testing.Furthermore, using the imaging device may permit the monitoring of theresponse of the bacterially-infected tissues to a variety of medicaltreatments, including the use of antibiotics and other therapies, suchas photodynamic therapy (PDT), hyperbaric oxygen therapy (HOT), lowlevel light therapy, or anti-Matrix Metalloproteinase (MMP). The devicemay be useful for visualization of bacterial biodistribution at thesurface as well as within the tissue depth of the wound, and also forsurrounding normal tissues. The device may thus be useful for indicatingthe spatial distribution of an infection.

Use of Device with Contrast Agents in Monitoring Wounds

The device may be used with exogenous contrast agents, for example thepro-drug aminolaevulinic acid (ALA) at a low dose. ALA may be topicallyadministered to the wound, and imaging may be performed 1-3 hours laterfor enhanced red fluorescence of wound bacteria.

The pro-drug aminolaevulinic acid (ALA) induces porphyrin formation inalmost all living cells. Many bacteria species exposed to ALA are ableto induce protoporphyrin IX (PpIX) fluorescence. The use of ultra-lowdose ALA may induce PpIX formation in the bacteria and hence mayincrease the red fluorescence emission, which may enhance thered-to-green fluorescence contrast of the bacteria imaged with thedevice. ALA is non-fluorescent by itself, but PpIX is fluorescent ataround 630 nm, 680 and 710 nm, with the 630 nm emission being thestrongest. The imaging device may then be used to image the green andred fluorescence from the wound and the surrounding tissues. The timeneeded to obtain significant/appreciable increase in red (e.g., peak at630 nm) fluorescence using the imaging device after the ALA (˜20 μg/mL)was applied to the wound ranges from 10-30 mins, but this time can beoptimized, and depends also on the ALA dose which can also be optimized.

Thus, a clinical operator can premix the ALA, which is usually providedcommercially in lyophilized form with physiological saline or other typeof commercially available cream/ointment/hydrogel/dressing etc., at agiven dose and administer the agent topically by spraying it, pouringit, or carefully applying the agent to the wound area prior to imaging.Approximately 10-30 mins afterwards, although this time may vary,fluorescence imaging may be performed in a dimly lit or dark room.Bacteria occult under white light and perhaps poorly autofluorescent mayappear as bright red fluorescent areas in and around the wound. Thefluorescence images may be used to direct targeted swabbing, biopsyand/or fine needle aspirates of the wound for bacterial culturing basedon the unique bacterial fluorescence signal, and this may be done atdifferent depths, for superficial and deep wounds.

The device may also be used in conjunction with exogenous ‘pro-drug’agents, including, but not limited to, ALA which is FDA approved forclinical therapeutic indications, to increase the endogenous productionof porphyrins in bacteria/microorganisms and thereby increase theintensities of unique ‘porphyrin’ fluorescence signals emanating fromthese bacteria to improve the detection sensitivity and specificity ofthe device. Thus, the device may be used to conveniently imagephotosensitizer-induced fluorescence (e.g., PpIX) in bacteria, growingin culture or in patients' wounds for subsequent image-guided targetedswabbing/biopsy or treatment, for example using photodynamic therapy(PDT) or hyperbaric oxygen therapy (HOT). The device when used with forexample consumable, commercially available fluorescence contrast agentshas the ability to increase the signal-to-background for sensitivedetection of bacteria, in and around wounds. It should be noted that ALAis commercially available.

In one example, the device was used to image live bacterial culture(staphylococcus aureus, grown on agar plates for 24 h prior to imaging)using violet/blue excitation light. After 30 mins of incubation ofstaphylococcus aureus ˜20 μg/mL of ALA at 37° C., a significant increasein red fluorescence from the bacteria was detected, compared with thosecolonies that did not receive any ALA. Thus, the device may exploit theuse of contrast agent strategies to increase the signal-to-backgroundfor sensitive detection of bacteria, in wounds for example. The timeneeded for the ALA to increase the PpIX fluorescence of bacteria inculture to significant levels was approximately 0.5 h which suggeststhat this approach may be clinically practical. Tests on simulatedbacterially-contaminated meat samples revealed similar results to thoseobtained from bacterial culture. Topical application of 0.2 μg/mL ALA byspraying onto wounds on pig skin resulted in a dramatic increase ofbacterial porphyrin red fluorescence contrast approximately 2 h afterALA administration. This demonstrates that the device may allow fordetection of bacterial contamination with fluorescence imaging withinthe wound sites and elsewhere on the skin surface, which was previouslyoccult under white light imaging.

Use with Exogenous Molecular-Targeted and Activated Imaging Agents

The availability of commercially available fluorescence molecularbacteriological detection and viability kits may offer another use forthe device in wound care. Such kits may be used to rapidlyquantitatively distinguish live and dead bacteria, even in a mixedpopulation containing a range of bacterial types. Conventionaldirect-count assays of bacterial viability are typically based onmetabolic characteristics or membrane integrity. However, methodsrelying on metabolic characteristics often only work for a limitedsubset of bacterial groups, and methods for assessing bacterial membraneintegrity commonly have high levels of background fluorescence. Bothtypes of determinations also suffer from being very sensitive to growthand staining conditions.

Suitable exogenous optical molecular targeting probes may be preparedusing commercially available fluorescence labeling kits, such as theAlexa Fluor active esters and kits (e.g., Zenon Antibody Labeling Kitsand or EnzChek Protease Assay Kits, Invitrogen) for labeling proteins,monoclonal antibodies, nucleic acids and oligonucleotides (Invitrogen).For example, these fluorescent dye bioconjugates cover the followingwavelength ranges: Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430,Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532,Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594,Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647,Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700 and Alexa Fluor 750dyes, where the number stated refers to the excitation wavelength of thedye. These kits may offer well-differentiated fluorescence emissionspectra, providing many options for multicolor fluorescence detectionand fluorescence resonance energy transfer, based on the appropriateselection of fluorescence emission filters with the imaging device. Thefluorescence dyes offer high absorbance at wavelengths of maximal outputof common excitation sources, they are bright and unusually photostablefluorescence of their bioconjugates, and offer good water solubility ofthe reactive dyes for ease of conjugation within the clinical exam roomand resistance of the conjugates to precipitation and aggregation. Thedyes' fluorescence spectra are insensitive to pH over a broad range,which makes them particularly useful for wound imaging, since wound pHcan vary. In addition, other commercial or non-commercial fluorescentagents exist which may be appropriate for biological imaging of woundsand may be combined with the described device, including fluorescentblood pooling agents and various wound-enzyme or protease activatedprobes from VisEn Medical (Boston, Mass., USA), for example.

These targeting fluorescent bioconjugates may be prepared using suchlabeling kits prior to the clinical exam of the wound using the imagingdevice in fluorescence mode, and may be stored in light-tight containersto avoid photobleaching. Such fluorescence bioconjugates may be preparedin solution at a known and appropriate concentration prior tofluorescence imaging of the wound using the device, and thenadministered/applied directly to the wound and surrounding normaltissues either topically (e.g., via aerosol/spray, lavage techniques),or given orally in a drink or systemically via intravenous injection.Such dyes may target specific biological components depending on thetargeting moiety, and may include: bacteria, fungi, yeast, spores,virus, microbes, parasites, exudates, pH, blood vessels, reducednicotinamide adenine dinucleotide (NADH), Flavin adenine dinucleotide(FAD), microorganisms, specific types of connective tissues (e.g.,collagens, elastin), tissue components, vascular endothelial growthfactor (VEGF), endothelial growth factor (EGF), epithelial growthfactor, epithelial cell membrane antigen (ECMA), hypoxia induciblefactor (HIF-1), carbonic anhydrase IX (CAIX), laminin, fibrin,fibronectin, fibroblast growth factor, transforming growth factors(TGF), fibroblast activation protein (FAP), enzymes (e.g., caspases,matrix metalloproteinases (MMPs), etc.), tissue inhibitors ofmetalloproteinases (e.g., TIMPs), nitric oxide synthase (NOS), inducibleand endothelial NOS, lysosomes in cells, macrophages, neutrophils,lymphocytes, hepatocyte growth factor (HGF), anti-neuropeptides, neutralendopeptidase (NEP), granulocyte-macrophage colony stimulating factor(GM-CSF), neutrophil elastases, cathepsins, arginases, fibroblasts,endothelial cells and keratinocytes, keratinocyte growth factor (KGF),macrophage inflammatory protein-2 (MIP-2), macrophage inflammatoryprotein-2 (MIP-2), and macrophage chemoattractant protein-1 (MCP-1),polymorphonuclear neutrophils (PMN) and macrophages, myofibroblasts,interleukin-1 (IL-1) and tumour necrosis factor (TNF), nitric oxide (NO)(Kit from Calbiochem, Model DAF-2 DA), and c-myc and beta-catenin,circulating endothelial progenitor cells (EPCs) from the bone marrow.Exogenous optical agents may include, but are not limited to, any of thefollowing: activated molecular beacons (e.g., targeted), nanoparticleshaving fluorescent agents (e.g., labeled on the surface and/orcontaining or carrying fluorescent agents), and scattering or absorbingnanoparticles (e.g., gold, silver).

The LIVE/DEAD BacLight™ Bacterial Viability Kits (from Invitrogen,Molecular Probes) assay utilizes mixtures of SYTO® 9 green fluorescentnucleic acid stain and the red fluorescent nucleic acid stain, propidiumiodide, although these fluorescent dyes may be exchanged for otherexisting or emerging fluorescent agents. These stains differ both intheir spectral characteristics and in their ability to penetrate healthybacterial cells. When used alone, the SYTO 9 stain labels bacteria withboth intact and damaged membranes. In contrast, propidium iodidepenetrates only bacteria with damaged membranes, competing with the SYTO9 stain for nucleic acid binding sites when both dyes are present. Whenmixed in recommended proportions, SYTO 9 stain and propidium iodideproduce green fluorescent staining of bacteria with intact cellmembranes and red fluorescent staining of bacteria with damagedmembranes. Thus, live bacteria with intact membranes fluoresce green,while dead bacteria with damaged membranes fluoresce red. The backgroundremains virtually non-fluorescent. Consequently, the ratio of green tored fluorescence intensities may provide a quantitative index ofbacterial viability.

Live and dead bacteria may be viewed separately or simultaneously by theimaging device with suitable optical filter sets. As well, similarfluorescence assay kits are available for Gram sign (i.e.,positive/negative) identification of bacteria, which is a usefulparameter in wound treatment planning, and may be used in conjunctionwith the imaging device. Such fluorescence agents are general andapplicable to most bacteria types, and may be used to determinebacterial viability and/or Gram sign either directly on/within the woundor on ex vivo swab- or tissue biopsy-derived culture samples obtainedfrom the wound site (e.g., superficially or at depth) for real-timequantitative assessment using the imaging device. Such fluorescencefluorescent agents may be prepared in solution in advance at a known andappropriate concentration prior to fluorescence imaging of the woundusing the device, and then administered/applied directly to the woundand surrounding normal tissues either topically (e.g., viaaerosol/spray, lavage techniques), or perhaps systemically viaintravenous injection. Imaging may then be performed accordingly after adefined time for the agents to react with the targets. A washing off ofunlabeled agents may be required prior to imaging with the device. Forthis, physiological saline may be used. Target-bound fluorescent agentmay remain within the wound and surrounding tissues for fluorescenceimaging.

Therefore, when used with fluorescent reporter systems the imagingdevice may provide a relatively rapid means of assessing bacterialviability following exposure to antimicrobial agents. The ability torepeatedly measure the same patients or animals may reduce variabilitywithin the treatment experiments and allowed equal or greater confidencein determining treatment efficacy. This non-invasive and portableimaging technology may reduce the number of animals used during suchstudies and has applications for the evaluation of test compounds duringdrug discovery.

A number of commercially available organic fluorophores have propertiesthat are dependent on hydrogen ion concentration, rendering them usefulas probes for measuring pH, and they typically have pH sensitiveUV/visible absorption properties. The majority of commercially availablepH sensitive fluorescent dyes employed in intracellular studies providea reduced fluorescent signal in acidic media or alternatively the pKa ofthe dye is outside the critical intracellular pH window of between 5-8pH units. However, other pH-sensitive fluorescent agents respond byincreasing their fluorescence intensities. For example,Invitrogen/Molecular Probes offers a variety of fluorescent pHindicators, their conjugates and other reagents for pH measurements inbiological systems. Among these are several probes with unique opticalresponse and specialized localization characteristics: for example,visible light-excitable SNARF pH indicators enable researchers todetermine intracellular pH in the physiological range usingdual-emission or dual-excitation ratiometric techniques, thus providinguseful tools for confocal laser-scanning microscopy and flow cytometry.LysoSensor probes, as well as indicators based on the Oregon Greenfluorophore, may be used to estimate the pH in a cell's acidicorganelles. There are also fluorescent pH indicators coupled to dextranswhich may be used. Following loading into cells, indicator dextrans maybe well retained, may not bind to cellular proteins and may have areduced tendency to compartmentalize. Again, such fluorescent agents maybe prepared in solution in advance at a known and appropriateconcentration prior to fluorescence imaging of the wound using thedevice, and then administered/applied directly to the wound andsurrounding normal tissues either topically (e.g., via aerosol/spray,lavage techniques), systemically or for example, via intravenousinjection, or orally.

EXAMPLES

Reference is now made to FIG. 24. As an example, the imaging device maybe used clinically to determine the healing status of a chronic woundand the success of wound debridement. For example, a typical foot ulcerin a person with diabetes is shown in the figure, with (i) thenonhealing edge (i.e., callus) containing ulcerogenic cells withmolecular markers indicative of healing impairment and (ii)phenotypically normal but physiologically impaired cells, which can bestimulated to heal. Despite a wound's appearance after debridement, itmay not be healing and may need to be evaluated for the presence ofspecific molecular markers of inhibition and/or hyperkeratotic tissue(e.g., c-myc and β-catenin). Using the imaging device in combinationwith exogenous fluorescently labeled molecular probes against suchmolecular targets, the clinician may be able to determine the in situexpression of molecular biomarkers. With the device, once a wound isdebrided, fluorescence imaging of the wound area and image analyses mayallow biopsy targeting for subsequent immunohistochemistry and this maydetermine whether the extent of debridement was sufficient. If theextent of debridement was insufficient, as shown in the lower leftdiagram, cells positive for c-myc (which appears green) and nuclearβ-catenin (which appears purple) may be found based on theirfluorescence, indicating the presence of ulcerogenic cells, which mayprevent the wound from healing properly and indicate that additionaldebridement is necessary. Lack of healing may also be demarcated by athicker epidermis, thicker cornified layer, and presence of nuclei inthe cornified layer. If the debridement was successful, as in the lowerright lower diagram, no staining for c-myc or β-catenin may be found,indicating an absence of ulcerogenic cells and successful debridement.These markers of inhibition may be useful, but the goal is actualhealing as defined by the appearance of new epithelium, decreased areaof the wound, and no drainage. This information may be collected usingthe fluorescence imaging device and stored electronically in thepatient's medical record, which may provide an objective analysiscoupled with pathology and microbiology reports. By comparing expectedhealing time with actual healing (i.e., healing progress) time using theimaging device, adaptive treatment strategies may be implemented on aper-patient basis.

FIG. 24B shows an example of the use of the device for imaging woundhealing of a pressure ulcer. a) White light image taken with the deviceof the right foot of a diabetic patient with a pressure ulcer is shown.b) Corresponding fluorescence image shows the bright red fluorescence ofbacteria (bacteriology results confirmed presence of heavy growth ofStaphylococcus aureus) which are invisible under standard white lightexamination (yellow arrows). Note the heavy growth of Staphylococcusaureus bacteria around the periphery of the non-healing wound (longyellow arrow). c-d) Show the spectrally-separated (unmixed)red-green-blue images of the raw fluorescence image in b), which areused to produce spectrally-encoded image maps of the green (e.g.collagen) and red (e.g. bacteria) fluorescence intensities calculatedusing mathematical algorithms and displayed in false color with colorscale. f-g) show examples of image-processing methods used enhance thecontrast of the endogenous bacterial autofluorescence signal bycalculating the red/green fluorescence intensity ratio to reveal thepresence and biodistribution of bacteria (red-orange-yellow) within andaround the open wound. These data illustrate the ability to use customor commercially-available image-analysis software to mathematicallyanalyze the fluorescence images obtained by the device and display themin a meaningful way for clinical use, and this may be done in real-time.(Scale bar 1 cm).

FIG. 24C shows an example of the use of the device for imaging a chronicnon-healing wound. a) White light image taken with the device of theleft breast of a female patient with Pyoderma gangrenosum, shows achronic non-healing wound (blue arrow) and a healed wound (red arrow).Bacteria typically cannot be visualized by standard white lightvisualization used in conventional clinical examination of the wounds.b) Corresponding fluorescence image of the same wounds (in this example,using 405 nm excitation, 500-550 nm emission (green), >600 nm emission(red)) is shown. Note that the non-healed wound appears dark coloredunder fluorescence (mainly due to blood absorption of the excitation andfluorescence emission light), while bacteria appear as punctuate brightred spots in the healed wound (red arrow). Under fluorescence, normalsurrounding skin appears cyan-green due to endogenous collagenfluorescence (405 nm excitation). By contrast, the non-healed wound(blue arrow) appears to have a band of very bright red fluorescencearound the wound border, confirmed with swab cultures (bacteriology) tocontain a heavy growth of Staphylococcus aureus (with few Gram positivebacilli and rare Gram positive cocci, confirmed by microscopy). c) Whitelight image of the healed wound in a), b) and d) correspondingfluorescence image showing bright red fluorescence from bacteria (pinkarrows), which are occult under white light. e) White light and f)corresponding fluorescence images of the non-healed breast wound. Notethat bacteria (Staphylococcus aureus) appear to be mainly localizedaround the edge/boundary of the wound (yellow arrow), while lessbacteria are located within the wound (X), determined by thebiodistribution of bacteria directly visualized using fluorescenceimaging, but invisible under white light (black arrow, e). (Scale bar incm).

FIG. 24D further illustrates imaging of a chronic non-healing woundusing an example of the imaging device. a) White light image taken withthe device of left breast of female patient with Pyoderma gangrenosum,showing chronic non-healing wound (blue arrow) and healed wound (bluearrow). Bacteria cannot be visualized by standard white lightvisualization used in clinical examination of the wounds. b)Corresponding fluorescence image of the same wounds (405 nm excitation,500-550 nm emission (green), >600 nm emission (red)). While the nippleappears to be normal under white without obvious contamination ofbacteria, fluorescence imaging shows the presence of bacteria emanatingfrom the nipple ducts. Swabs of the nipple showed bacteria wereStaphylococcus epidermidis (Occasional growth found on culture). (Scalebar in cm)

FIG. 24E shows a central area and border of a chronic non-healing woundimaged using the imaging device. a) White light image taken with thedevice of left breast of female patient with Pyoderma gangrenosum,showing the central area and border of a chronic non-healing wound. a)White light and b) corresponding fluorescence images of the non-healedbreast wound (405 nm excitation, 500-550 nm emission (green), >600 nmemission (red)). Note that bacteria (Staphylococcus aureus; shown bybacterial swabbing) appear to be mainly localized around theedge/boundary of the wound, while less bacteria are located within thewound (X), determined by the biodistribution of bacteria directlyvisualized using fluorescence imaging, but invisible under white light.(Scale bar in cm).

FIG. 24F shows further images of a chronic non-healing wound using theimaging device. a) White light image taken with the device of leftbreast of female patient with Pyoderma gangrenosum, showing chronicnon-healing wound. Bacteria cannot be visualized by standard white lightvisualization used in clinical examination of the wounds. b)Corresponding fluorescence image of the same wound (405 nm excitation,500-550 nm emission (green), >600 nm emission (red)). Fluorescenceimaging shows the presence of bacteria around the wound edge/borderpre-cleaning (b) and post-cleaning (c). In this example, cleaninginvolved the use of standard gauze and phosphate buffered saline to wipethe surface the wound (within and without) for 5 minutes. Aftercleaning, the red fluorescence of the bacteria is appreciably decreasedindicating that some of the red fluorescent bacteria may reside belowthe tissue surface around the edge of the wound. Small amounts ofbacteria (red fluorescent) remained within the wound center aftercleaning. This illustrates the use of the imaging device to monitor theeffects of wound cleaning in real-time. As an additional example, d)shows a white light image of a chronic non-healing wound in the samepatient located on the left calf. e) Shows the correspondingfluorescence images pre-cleaning (e) and post-cleaning (f). Swabbing ofthe central area of the wound revealed the occasional growth ofStaphylococcus aureus, with a heavy growth of Staphylococcus aureus atthe edge (yellow arrow). Cleaning resulted in a reduction of thefluorescent bacteria (Staphylococcus aureus) on the wound surface asdetermined using the handheld optical imaging device. The use of theimaging device resulted in the real-time detection of white light-occultbacteria and this allowed an alteration in the way the patient wastreated such that, following fluorescence imaging, wounds andsurrounding (bacteria contaminated) were either re-cleaned thoroughly orcleaned for the first time because of de novo detection of bacteria.Also, note the use of a disposable adhesive measurement-calibration‘strip’ for aiding in imaging-focusing and this “strip” may be adheredto any part of the body surface (e.g., near a wound) to allow woundspatial measurements. The calibration strip may also be distinctlyfluorescent and may be used to add patient-specific information to theimages, including the use of multiple exogenous fluorescent dyes for“barcoding” purposes—the information of which can be integrated directlyinto the fluorescence images of wounds. (Scale bar in cm).

FIG. 24G illustrates use of the imaging device for monitoring woundhealing over time. The imaging device is used for tracking changes inthe healing status and bacterial biodistribution (e.g. contamination) ofa non-healing chronic wound from the left breast of female patient withPyoderma gangrenosum. White light images (a-m) and correspondingfluorescence images of the (b-n) healed wound and of the (c-o) chronicnon-healing wound are shown over the course of six weeks. (405 nmexcitation, 500-550 nm emission (green), >600 nm emission (red)), takenusing the imaging device under both white light and fluorescence modes.In b-n), the presence of small bright red fluorescence bacterialcolonies are detected (yellow arrows), and their localization changesover time within the healed wound. Bacterial swabs confirmed that nobacteria were detected on microscopy and no bacterial growth wasobserved in culture. In c-o), by contrast, the non-healed wound has aband of very bright red fluorescence around the wound border, confirmedwith swab cultures (bacteriology) to contain a heavy growth ofStaphylococcus aureus (with few Gram positive bacilli and rare Grampositive cocci, confirmed by microscopy), which changes inbiodistribution over time (i.e., c-o). These data demonstrate that theimaging device may yield real-time biological and molecular informationas well as be used to monitor morphological and molecular changes inwounds over time.

FIG. 24H shows another example of the use of the device for monitoringwound status over time. The imaging device is used tracking changes inthe healing status and bacterial biodistribution (e.g. contamination) ofa wound from the left calf of 21 year old female patient with Pyodermagangrenosum. White light images (a-i) and corresponding fluorescenceimages of a (b-j) wound being treated using hyperbaric oxygen therapy(HOT) are shown over the course of six weeks. (Fluorescence parameters:405 nm excitation, 500-550 nm emission (green), >600 nm emission (red)).a-i) White light images reveal distinct macroscopic changes in the woundas it heals, indicated by the reduction in size over time (e.g. closure)from week 1 (˜2 cm long diameter diameter) through to week 6 (˜0.75 cmlong axis diameter). In b-j), the real-time fluorescence imaging ofendogenous bacterial fluorescence (autofluorescence) in and around thewound can be tracked over time, and correlated with the white lightimages and wound closure measurements (a-i). b) shows a distinct greenband of fluorescence at the immediate boundary of the wound (yellowarrow; shown to be contaminated heavy growth of Staphylococcus aureus),and this band changes over time as the wound heals. Red fluorescencebacteria are also seen further away from the wound (orange arrow), andtheir biodistribution changes over time (b-j). Thewound-to-periwound-to-normal tissue boundaries can be seen clearly byfluorescence in image j). Connective tissue (in this example, collagens)in normal skin appear as pale green fluorescence (j) and connectivetissue remodeling during wound healing can be monitored over time,during various wound treatments including, as is the case here,hyperbaric oxygen therapy of chronic wounds.

FIG. 24I illustrates use of the imaging device for targeting bacterialswabs during routine wound assessment in the clinic. Under fluorescenceimaging, the swab can be directed or targeted to specific areas ofbacterial contamination/infection using fluorescence image-guidance inreal-time. This may decrease the potential for contamination ofnon-infected tissues by reducing the spread of bacteria during routineswabbing procedures, which may be a problem in conventional woundswabbing methods. Swab results from this sample were determined to beStaphylococcus aureus (with few Gram positive bacilli and rare Grampositive cocci, confirmed by microscopy).

FIG. 24J shows an example of the co-registration of a) white light andb) corresponding fluorescence images made with the imaging device in apatient with diabetes-associated non-healing foot ulcers. Using anon-contact temperature measuring probe (inset in a) with cross-lasersighting, direct temperature measurements were made on normal skin(yellow “3 and 4”) and within the foot ulcers (yellow “1 and 2”)(infected with Pseudomonas aeruginosa, as confirmed by bacteriologicalculture), indicating the ability to add temperature-based information tothe wound assessment during the clinical examination. Infected woundshave elevated temperatures, as seen by the average 34.45° C. in theinfected wounds compared with the 30.75° C. on the normal skin surface,and these data illustrate the possibility of multimodality measurementswhich include white light, fluorescence and thermal information forwound health/infectious assessment in real-time. Note that bothnon-healing wounds on this patient's right foot contained heavy growthof Pseudomonas aeruginosa (in addition to Gram positive cocci and Gramnegative bacilli), which in this example appear as bright greenfluorescent areas within the wound (b).

FIG. 24K shows an example of the use of the imaging device formonitoring a pressure ulcer. a) White light image taken with the imagingdevice of the right foot of a Caucasian diabetic patient with a pressureulcer is shown. b) Corresponding fluorescence image shows the bright redfluorescence of bacteria (bacteriology results confirmed presence ofheavy growth of Staphylococcus aureus) which are invisible understandard white light examination (yellow arrows). Dead skin appears as awhite/pale light green color (white arrows). Note the heavy growth ofStaphylococcus aureus bacteria around the periphery of the non-healingopen wounds (yellow arrows). c) Shows the fluorescence imaging of atopically applied silver antimicrobial dressing. The imaging device maybe used to detect the endogenous fluorescence signal from advanced woundcare products (e.g., hydrogels, wound dressings, etc.) or thefluorescence signals from such products which have been prepared with afluorescent dye with an emission wavelength within the detectionsensitivity of the imaging detector on the device. The device may beused for image-guided delivery/application of advanced wound caretreatment products and to subsequently monitor their distribution andclearance over time.

FIG. 24L shows an example of the use of the device for monitoring apressure ulcer. a) White light image taken with the device of the rightfoot of a Caucasian diabetic patient with a pressure ulcer. b)Corresponding fluorescence image shows the bright red fluorescent areaof bacteria (bacteriology results confirmed presence of heavy growth ofStaphylococcus aureus, SA) at the wound edge and bright greenfluorescent bacteria (bacteriology results confirmed presence of heavygrowth of Pseudomonas aeruginosa, PA) which are both invisible understandard white light examination. c) Fluorescence spectroscopy taken ofthe wound revealed unique spectral differences between these twobacterial species: SA has a characteristic red (about 630 nm)autofluorescence emission peak, while PA lacks the red fluorescence buthas a strong green autofluorescence peak at around 480 nm.

FIG. 24M shows an example of the use of the device for monitoring achronic non-healing wound. a) White light image taken with the imagingdevice of chronic non-healing wounds in 44 year old black male patientwith Type II diabetes is shown. Bacteria cannot be visualized bystandard white light visualization (a-g) used in conventional clinicalexamination of the wounds. b-h) Corresponding fluorescence image of thesame wounds (405 nm excitation, 500-550 nm emission (green), >600 nmemission (red)). This patient presented with multiple open non-healingwounds. Swab cultures taken from each wound area using the fluorescenceimage-guidance revealed the heavy growths of Pseudomonas aeruginosa(yellow arrow) which appear bright green fluorescent, and Serratiamarcescens (circles) which appear red fluorescent. (Scale bar in cm).

FIG. 24N is a schematic diagram illustrating an example of a use of“calibration” targets, which may be custom-designed, multi-purpose,and/or disposable, for use during wound imaging with the imaging device.The strip, which in this example is adhesive, may contain a combinationof one or more of: spatial measurement tools (e.g., length scale),information barcode for integrating patient-specific medicalinformation, and impregnated concentration-gradients of fluorescent dyesfor real-time fluorescence image calibration during imaging. For thelatter, multiple concentrations of various exogenous fluorescent dyes orother fluorescence agents (e.g., quantum dots) may be used formultiplexed fluorescence intensity calibration, for example when morethan one exogenous fluorescently-labeled probe is used fortissue/cell/molecular-targeted molecular imaging of wounds in vivo.

FIG. 24O shows an example of the use of an embodiment of the imagingdevice for monitoring bacteria, for example for monitoring a treatmentresponse. a) Fluorescence microscopy image of a live/dead bacteria stainsold by Invitrogen Corp. (i.e., BacLight product). b) Fluorescencemicroscopy image of a Gram staining bacteria labeling stain sold byInvitrogen Corp. Using the imaging device (c) with such products, live(green) and dead (red) bacteria (e) may be distinguished in real-time exvivo (e.g., on the swab or tissue biopsy) following bacterial swabbingof a wound, or other body surface, for example, in the swabbing of theoral buccal cheek, as in d). This real-time bacterial Gram staining orlive/dead image-based assessment may be useful for real-time orrelatively rapid bacteriology results that may be used for refiningtreatments, such as antibiotic or other disinfective treatments, or formonitoring treatment response.

FIG. 24P shows an example of the use of the device used for imaging oftoe nail infection. a) White light and b) corresponding autofluorescenceof the right toe of a subject demonstrating the enhanced contrast of theinfection that fluorescence imaging provides compared to white lightvisualization (405 nm excitation, 500-550 nm emission (green), >600 nmemission (red)).

FIG. 24Q shows and example of imaging using the device for monitoringthe response of meat-infected with bacteria to a disinfectant (e.g.hydrogen peroxide (Virox5™)). a) An ex vivo porcine tissue sample wasprepared in Petri dishes and contaminated with Staphylococcus aureusprior to topical administration of b) Virox5™ and fluorescence imaging(with handheld device), c). Breakdown of the tissue begins to occurrapidly, caused by the disinfectant, while a change in the fluorescencecharacteristics of the bacteria becomes apparent (e.g. red fluorescencecolor begins to change to orange fluorescence color, as seen in d),especially after gentle agitation of the sample and over time, hereabout 5 minutes incubation with the Virox5™ solution. These data suggestthe use of the device for monitoring bacterial disinfection, for examplein clinical and non-clinical settings (405 nm excitation; 490-550 nmand >600 nm emission).

In addition to fluorescence-enhancing pro-drugs, advances in medicinehave enabled widespread use of fluorescent biomarkers to diagnosedisease on a molecular level. The accurate measurement of thefluorescent biomarker signal in biological tissues may be a criticalparameter towards gaining biomolecular information about diseaseprogression and treatment response, but has historically posed asignificant challenge. To date, this type of advanced molecular imaginghas not been reported for wound care.

The device described herein may also be used in combination withfluorescent, light-scattering, or light-absorbing exogenous fluorescencecontrast agents that can be used passively and/or targeted to unique andspecific molecular targets within the wound to improve the detection anddiagnosis of wound infection. These targets may be any biological and/ormolecular component in the wound or normal surrounding tissues that havea known detection and/or diagnostic value (e.g., normal tissue and woundbiomarkers). All exogenous agents may be delivered to the wound eithertopically and/or systemically, and may include, but are not limited to,any exogenous agent/drug (e.g., encapsulated liposomes, beads or otherbiocompatible carrier agents) that can be coupled/conjugated with anappropriate wavelength-selected fluorescent/scattering moiety (e.g.,organic fluorescent dyes, quantum dots and other fluorescentsemi-conductor nano-particles, colloidal metals (e.g., gold, silver,etc.)). Fluorescent and/or light scattering agents/probes, and/orchromogenic (i.e., absorption) agents/dyes may be prepared usingstandard bioconjugation techniques to include moieties for targetingspecific biomarkers. Such moieties may include monoclonal antibodies(e.g., whole and/or fragments), and other tissue-specific moieties(including, but not limited to, peptides, oligomers, aptamers,receptor-binding molecules, enzyme inhibitors, toxins, etc.). The devicemay also be used for imaging in situ activatable promoter-controlledexpression of light generating proteins in preclinical wound models.Furthermore, wound infections may also be detected using the imagingdevice and then treated using photothermal therapies, such aslight-absorbing gold nanoparticles conjugated with specific antibodieswhich specifically target bacteria.

FIG. 24R shows an example of use of the imaging device used for imagingof fluorescent dyes/probes/agents on biological tissues. a) White lightimaging of a piece of meat (ex vivo) does not reveal the presence of afluorescent dye, whereas in b) the device allows accurate fluorescencedetection and monitoring of the biodistribution of the fluorescent dye.Although shown for ex vivo tissue, these capabilities may be translatedto in vivo applications including but not limited to, for example,imaging the biodistribution of fluorescent photosensitizers withintissues for photodynamic therapy (PDT) of wounds, cancer, infection, orother diseases. White light imaging may provide anatomical context forthe fluorescence imaging. These capabilities may also be used to monitorphotobleaching of fluorescent agents (including photosensitizers) aswell as for image-guided delivery of multiple PDT treatments (405 nmexcitation, 500-550 nm emission (green), >600 nm emission (red)). Thedevice may provide for monitoring of pharmocokinetics, biodistribution,and/or photobleaching in PDT. Similarly, the device may be useful formonitoring of low level light therapies.

The device may also be used with other molecular-sensing agents, such as‘molecular beacons’ or “smart probes”, which produce fluorescence onlyin the presence of unique and specific biological targets (e.g., enzymesassociated with wound health). Such probes may be useful for identifyingspecific bacterial species or GRAM signing, for example. For example,cutaneous wound healing is a highly complex process involving fiveoverlapping phases (inflammation, granulation tissue formation,epithelialization, matrix production, and remodeling) associated with anumber of migratory and remodeling events that are believed to requirethe action of matrix metalloproteinases (MMPs) and their inhibitors,TIMPs. In vivo analyses of human acute and chronic wounds as well as ofa variety of different wound healing models have implicated a functionalrole of MMPs and TIMPs during normal wound repair, whereas deregulationof their activity is thought to contribute to impaired wound healing.Degradation of extracellular matrices is needed to remove damaged tissueand provisional matrices and to permit vessel formation andre-epithelialization. In contrast, in chronic or non-healing woundsover-expression of proteinases in their inactive form is thought tocontribute to the underlying pathology and to inhibit normal tissuerepair processes. Molecular beacons are activatable fluorescentreporters that use the fluorescence resonance energy transfer (FRET)principle to control fluorescence emission in response to specificbiological stimuli. They usually comprise a disease-specific linker thatbrings a quencher close to a fluorophore to silence its fluorescence.Upon specific linker-target interactions (e.g., nucleic acidhybridization, protease-specific peptide cleavage,phospholipase-specific phospholipids cleavage), the quencher is removedfrom the vicinity of the fluorophore to restore its fluorescence. Thesesmart probes may offer several orders of magnitude sensitivity thantargeted probes because of the built-in high degree of signalamplification from nonfluorescent to highly fluorescent. Depending ontheir specific linker-target interactions, they may also be capable ofinterrogating specific molecular abnormality at the protein or geneexpression levels. Because of these advantages, the smart probes havebeen recently hailed as “a quantum leap” over traditional probes forearly cancer detection. Such exogenous agents may be used, for example,for relatively rapid, non-invasive, sensitive and specific opticaldetection of wound infections, to identify specificbacterial/microorganism species present and in situ microbial diagnosis,to monitor the health status of the wound, and to report in real-time onthe effectiveness of treatment and care.

In addition, when used in combination with exogenous optical agents, thedevice may be used to identity patients minimally responsive to variousestablished and experimental treatments, enabling rapid non-invasive ornon-contact visual quantitative assessment of treatment response to maketimely changes in therapy in order to optimize treatment outcomes.

Furthermore, real-time monitoring of antimicrobial effects in vitro andwithin animal model test systems using the imaging device may enhancebasic understanding of the action of antibiotics and facilitate uniquestudies of disease in vivo.

EXAMPLES

FIG. 5 shows an example of the device being used for non-invasiveautofluorescence detection of collagen and varies bacterial species onthe skin surface of a pig meat sample. In contrast to white lightimaging, autofluorescence imaging was able to detect the presence ofseveral bacterial species 24 h after they were topically applied tosmall incisions made in the skin (i.e., streptococcus pyogenes, serratiamarcescens, staphylococcus aureus, staphylococcus epidermidis,escherichia coli, and pseudomonas aeruginosa). a) shows white lightimages of pig meat used for testing. Several bacterial species wereapplied to small incisions made in the skin at Day 0, and were labelledas follows: 1) streptococcus pyogenes, 2) serratia marcescens, 3)staphylococcus aureus, 4) staphylococcus epidermidis, 5) escherichiacoli, and 6) pseudomonas aeruginosa. The imaging device was used todetect collagen and bacterial autofluorescence over time. Connectivetissue fluorescence was intense and easily detected as well. Somebacterial species (e.g., pseudomonas aeruginosa) produces significantgreen autofluorescence (450-505 nm) which saturated the device's camera.b) shows autofluorescence image at Day 0, magnified in c).

The device was also able to detect spreading of the bacteria over thesurface of the meat over time. d) shows an image at Day 1, and f) showsan image at Day 3, as the meat sample was maintained at 37° C. Redfluorescence can be seen in some of the wound sites (5, 6) in c). Asshown in d) and magnified in e), after 24 h, the device detects adramatic increase in bacterial autofluorescence from wound site 5)escherichia coli and 6) pseudomonas aeruginosa, with the latterproducing significant green and red autofluorescence. c) and e) show thedevice detecting fluorescence using a dual band (450-505 nm green and590-650 nm) on the left and a single band filter (635+/−10 nm) on theright, of the wound surface. As shown in f), by Day 3, the devicedetects the significant increase in bacterial autofluorescence (in greenand red) from the other wound sites, as well as the bacterialcontamination (indicated by the arrow in f) on the styrofoam containerin which the meat sample was kept. The device was also able to detectspreading of the bacteria over the surface of the meat. Thisdemonstrates the real-time detection of bacterial species on simulatedwounds, the growth of those bacteria over time, and the capability ofthe device to provide longitudinal monitoring of bacterial growth inwounds. The device may provide critical information on thebiodistribution of the bacteria on the wound surface which may be usefulfor targeting bacterial swabbing and tissue biopsies. Note, in d) andf), the intense green fluorescence signal from endogenous collagen atthe edge of the pig meat sample.

This example demonstrates the use of the device for real-time detectionof biological changes in connective tissue and bacterial growth based onautofluorescence alone, suggesting a practical capability of the deviceto provide longitudinal monitoring of bacterial growth in wounds.

Reference is now made to FIG. 6, which shows examples of the device usedfor autofluorescence detection of connective tissues (e.g., collagen,elastin) and bacteria on the muscle surface of a pig meat sample. a)shows that white light image of pig meat used for testing shows noobvious signs of bacterial/microbial contamination or spoilage. However,as seen in b), imaging of the same area with the device underblue/violet light excitation revealed a bright red fluorescent area ofthe muscle indicating the potential for bacterial contamination comparedwith the adjacent side of muscle. Extremely bright greenautofluorescence of collagen can also be seen at the edge of the skin.In c), the device was used to surgically interrogate suspicious redfluorescence further to provide a targeted biopsy for subsequentpathology or bacteriology. Note also the capability of the device todetect by fluorescence the contamination (arrow) of the surgicalinstrument (e.g., forceps) during surgery. In d), the device was used totarget the collection of fluorescence spectroscopy using a fibre opticprobe of an area suspected to be infected by bacteria (inset shows thedevice being used to target the spectroscopy probe in the same area ofred fluorescent muscle in b, c). e) shows an example of the device beingused to detect contamination by various thin films of bacteria on thesurface of the Styrofoam container on which the meat sample was kept.Autofluorescence of the bacteria appears as streaks of green and redfluorescence under violet/blue excitation light from the variousbacterial species previously applied to the meat. Thus, the device iscapable of detecting bacteria on non-biological surfaces where they areoccult under standard white light viewing (as in a).

In addition to detection of bacteria in wounds and on the skin surface,the device was also able to identify suspicious areas of muscle tissue,which may then be interrogated further by surgery or targeted biopsy forpathological verification, or by other optical means such asfluorescence spectroscopy using a fiber optic probe. Also, it detectedcontamination by various bacteria on the surface of the Styrofoamcontainer on which the meat sample was kept. Autofluorescence of thebacteria appears as streaks of green and red fluorescence underviolet/blue excitation light from the various bacterial speciespreviously applied to the meat.

In order to determine the autofluorescence characteristics of bacteriagrowing in culture and in the simulated skin wounds,hyperspectral/multispectral fluorescence imaging was used toquantitatively measure the fluorescence intensity spectra from thebacteria under violet/blue light excitation. Reference is now made toFIG. 7. In FIG. 7, the device was used to detect fluorescence frombacteria growing in agar plates and on the surface of a simulated woundon pig meat, as discussed above for FIGS. 4 and 5. Bacterialautofluorescence was detected in the green and red wavelength rangesusing the device in the culture (a) and meat samples (d).Hyperspectral/multispectral imaging was used to image the bacteria (E.coli) in culture (b) and to measure the quantitative fluorescenceintensity spectra from the bacteria (red line-porphyrins,green-cytoplasm, blue-agar background) (c). The red arrow shows the 635nm peak of porphyrin fluorescence detected in the bacteria.Hyperspectral/multispectral imaging also confirmed the strong greenfluorescence (*, right square in d) from P. aeruginosa (with littleporphyrin fluorescence, yellow line in f) compared to E. coli (leftsquare in d) where significant porphyrin red fluorescence was detected.e)and g) show the color-coded hyperspectral/multispectral imagescorresponding to P. aeruginosa and E. coli, respectively, from the meatsurface after 2 days of growth (incubated at 37° C.); and f) and h) showthe corresponding color-coded fluorescence spectroscopy. In i),excitation-emission matrices (EEM) were also measured for the variousbacterial species in solution, demonstrating the ability to select theoptimum excitation and emission wavelength bandwidths for use withoptical filters in the imaging device. The EEM for E. coli shows stronggreen fluorescence as well as significant red fluorescence fromendogenous bacterial porphyrins (arrow).

This example shows that bacteria emit green and red autofluorescence,with some species (e.g., pseudomonas aeruginosa) producing more of theformer. Escherichia coli produced significant red autofluorescence fromendogenous porphyrins. Such intrinsic spectral differences betweenbacterial species are significant because it may provide a means ofdifferentiating between different bacterial species usingautofluorescence alone. Excitation-emission matrices (EEMs) were alsomeasured for each of the bacterial species used in these pilot studies,which confirmed that under violet/blue light excitation, all speciesproduced significant green and/or red fluorescence, the latter beingproduced by porphyrins. Spectral information derived fromexcitation-emission matrices may aid in optimizing the selection ofexcitation and emission wavelength bandwidths for use with opticalfilters in the imaging device to permit inter-bacterial speciesdifferentiating ex vivo and in vivo. In this way, the device may be usedto detect subtle changes in the presence and amount of endogenousconnective tissues (e.g. collagens and elastins) as well as bacteriaand/or other microorganisms, such as yeast, fungus and mold withinwounds and surrounding normal tissues, based on unique autofluorescencesignatures of these biological components.

In addition to fluorescence-enhancing pro-drugs, advances in medicinehave enabled widespread use of fluorescent biomarkers to diagnosedisease on a molecular level. The accurate measurement of thefluorescent biomarker signal in biological tissues may be a criticalparameter towards gaining biomolecular information about diseaseprogression and treatment response, but has historically posed asignificant challenge. To date, this type of advanced molecular imaginghas not been reported for wound care. With the use of the devicedescribed here, imaging and monitoring of such biomarkers for diagnosispurposes may be possible.

Imaging of Wound Models using Exogenous Contrast Agents

When used to assess wounds, tissue autofluorescence imaging may detectrelative changes in connective tissue remodeling during wound healing aswell as the early presence of bacteria either contaminating, colonizingand/or infecting wounds (including, but not limited to,bacterially-induced production of wound exudate and inflammation). Whenmost wounds are illuminated by violet/blue light, endogenous tissues inthe connective tissue matrix (e.g., collagen and elastin) emit acharacteristic strong green fluorescent signal, while endogenousbacteria emit a unique red fluorescence signal due to the production ofendogenous porphyrins. These bacteria include, but are not limited to,common species typically found at wound sites (e.g., staphylococcus,streptococcus, e. coli, and pseudomonas species). By usingautofluorescence, critical wound information is obtained in real-time toprovide a means of early detection of key biological determinants ofwound health status, which may aid in stratifying patients for optimizedwound care and treatment.

The pro-drug aminolaevulinic acid (ALA) induces porphyrin formation inalmost all living cells. Many bacteria species exposed to ALA are ableto induce protoporphyrin IX (PpIX) fluorescence [Dietel et al., (2007).Journal of Photochemistry and Photobiology B: Biology. 86: 77-86]. Theuse of ultra-low dose ALA to induce PpIX formation in the bacteria andhence increase the red fluorescence emission was investigated, in orderto enhance the red-to-green fluorescence contrast of the bacteria withthe imaging device. The device was used to image live bacterial culture(staphylococcus aureus, grown on agar plates for 24 h prior to imaging)using violet/blue excitation light, as seen in FIG. 8, whichdemonstrates the device being used in a bacteriology/culture laboratory.

In a), the device was used to image live bacterial culture(staphylococcus aureus, grown on agar plates for 24 h prior to imaging)under white light (circles). In b), violet/blue excitation light revealsthe bacterial red autofluorescence, which is discernable from thebackground weak green autofluorescence from the agar growth medium. Inc), to increase the red-to-green fluorescence contrast of thestaphylococcus aureus against the background agar, an ultra-low dose(˜20 μg/mL) of the photosensitizer aminolevulinic acid (ALA, inphosphate buffered saline) commonly used in photodynamic therapy (PDT)was added topically to some of the colonies in the agar plate (noted as‘ALA+’ in the circles), while the rest of the agar plate wasALA-negative. After 30 mins of incubation at 37° C., the device wasagain used to image the agar plate under violet/blue light excitation,thus revealing a significant increase in red fluorescence (fromALA-induced protoporphyrin IX, PpIX) from the staphylococcus aureusbacteria, compared with those colonies (square) that did not receive anyALA. Comparing b) with c) shows that the addition of ALA may bebeneficial for increased bacterial fluorescence. d) shows the RBG imagefrom c) with the green fluorescence from the agar plate removed, thusrevealing the increased red bacterial fluorescence in the s. aureuscolonies treated with ALA. This demonstrates the ability of the deviceto exploit the use of contrast agent strategies to increase thesignal-to-background for sensitive detection of bacteria, in wounds forexample. The time needed for the ALA to increase the PpIX fluorescenceto detectable levels was 30 mins which suggests that this technicalapproach may also be clinically practical. Furthermore, this alsodemonstrates that the device may be used to conveniently imagephotosensitizer fluorescence (e.g., PpIX) in bacteria, growing inculture or in patients' wounds for subsequent treatment using PDT.

After 30 mins of incubation of staphylococcus aureus ˜20 μg/mL of ALA at37° C., a significant increase in red fluorescence from the bacteria wasdetected, compared with those colonies (square) that did not receive anyALA. This demonstrates the ability of the device to exploit the use ofcontrast agent strategies to increase the signal-to-background forsensitive detection of bacteria, in wounds for example. The time neededfor the ALA to increase the PpIX fluorescence of bacteria in culture tosignificant levels was approximately 0.5 h which suggests that thistechnical approach may also be clinically practical. Tests on simulatedbacterially-contaminated meat samples revealed similar results to thoseobtained from bacterial culture. Topical application of 0.2 μg/mL ALA byspraying onto wounds on pig skin resulted in a dramatic increase ofbacterial porphyrin red fluorescence contrast approximately 2 h afterALA administration. This may allow detection of bacterial contaminationwith fluorescence imaging within the wound sites and elsewhere on theskin surface, which was previously occult under white light imaging, asdemonstrated with reference to FIGS. 9 and 10.

FIG. 9 shows examples of use of the device for autofluorescencedetection of connective tissues and varies bacterial species on the skinsurface of a pig meat sample. To determine if the intensity of thebacterial fluorescence may be enhanced for imaging with the device, thenon-toxic pro-drug aminolevulinic acid (ALA) (˜0.2 mg/mL PBS) wasapplied topically to the skin surface by spraying using a commonatomizer bottle. The meat sample was then placed in a light tightincubator at 37° C. for approximately 3-4 h until white light andfluorescence imaging was performed using the imaging device.

Referring to FIG. 9, a) shows white light images of pig meat used fortesting. In b), several bacterial species were applied to smallincisions made in the skin [(1) streptococcus pyogenes, 2) serratiamarcescens, 3) staphylococcus aureus, 4) staphylococcus epidermidis, 5)escherichia coli, and 6) pseudomonas aeruginosa)]. Under violet/blueexcitation light, the device shows bacterial autofluorescence (green andred fluorescence in the wound sites). The presence of endogenousporphyrin red fluorescence can be seen in other areas of the skinsurface as well (red arrow). Bright collagen fluorescence can also beseen at the edge of the sample (blue arrow). Bacteria on the surface ofthe styrofoam container holding the meat sample, also are detected byautofluorescence with the device, but are occult under white light (leftpanel). This indicates that the device may be used for detecting andimaging of the presence of bacteria or microbes and other pathogens on avariety of surfaces, materials, instruments (e.g., surgical instruments)in hospitals, chronic care facilities, old age homes, and other healthcare settings where contamination may be the leading source ofinfection. The device may be used in conjunction with standarddetection, identification and enumeration of indicator organisms andpathogens strategies.

In c), the non-toxic pro-drug aminolevulinic acid (ALA) (0.2 mg/mL) wasapplied topically to the skin surface in order to determine if bacterialfluorescence may be enhanced. The result, approximately 1 h after ALAadministration, was a dramatic increase in bacterial porphyrinfluorescence (bright red fluorescence) both on the skin tissue and woundsites, as well as on the surface of the styrofoam container on which themeat sample was kept (arrows). This illustrates the possibilities forbiopsytargeting by fluorescence image-guidance, and the use of thedevice for detection and subsequent treatment of infected areas usingPDT, for example.

FIG. 10 shows examples of the use of the device for fluorescencecontrast-enhanced detection of bacterial infection in a pig meat sample.a) shows white light image of the pig meat. Several bacterial specieswere applied to small incisions made in the skin (arrow). In b), thenon-toxic pro-drug aminolevulinic acid (ALA) (0.2 μg/mL) was appliedtopically to the skin surface by spraying using an common atomizerbottle and the imaging device was used to image the resultingALA-induced protoporphyrin IX (PpIX) red fluorescence. Images of theskin surface (˜2 h after ALA administration) using violet/blue light(405 nm), resulted in a dramatic increase of bacterial porphyrin redfluorescence contrast indicating the detection of the presence ofbacterial contamination with fluorescence imaging within the simulatedsurgical wound incisions (arrows) and elsewhere on the skin surface,which was previously occult under white light imaging (circle in a andb). Note that some areas of the skin surface which were not exposed tooxygen because the sample was placed ‘skin down’ in the container do notemit bright red fluorescence, possibly due to the suspected dependenceon oxygen for bacterial production of PpIX. Some bacteria produce abright green autofluorescence signals which is also detected by thedevice. In c), in another pig meat sample, bacteria occult under whitelight imaging (circle) are easily detected using autofluorescenceimaging alone (inset). However, as shown in d) the topical applicationof low dose ALA caused a dramatic increase in bacterial fluorescenceafter 2 h, demonstrating the utility of exogenous pro-drugs asfluorescence imaging contrast enhancing agents for improved detection ofbacterial contamination. Note the bright green autofluorescence ofendogenous collagen and elastins in the connective tissues in thesample. In e) and f), ALA-induced fluorescence allowed detection ofoccult bacteria on the skin surface (circles) offering the possibilityof image-guided biopsy-targeting, and use of the device for detectionand subsequent treatment of infected areas using PDT, for example.

The device may also be used in conjunction with exogenous ‘pro-drug’agents, including, but not limited to, ALA which is FDA approved forclinical therapeutic indications, to increase the endogenous productionof porphyrins in bacteria/microorganisms and thereby increase theintensities of unique ‘porphyrin’ fluorescence signals emanating fromthese bacteria to improve the detection sensitivity and specificity ofthe device. Thus, the device may be used to conveniently imagephotosensitizer-induced fluorescence (e.g., PpIX) in bacteria, growingin culture or in patients' wounds for subsequent image-guided targetedswabbing or biopsy, or treatment using photodynamic therapy (PDT) [Joriet al. Lasers Surg Med. 2006 June; 38(5):468-81; Dougherty et al. (1998)J. Natl. Cancer Inst. 90, 889-905; Carruth (1998) Int. J. Clin. Pract.52, 39-42; Bissonnette et al. (1997) Dermatol. Clin. 15, 507-519]. PDTmay provide an adjunct to current antibiotic treatment or an alternativewhere antibiotics no longer are working (e.g., drug-resistant strains).The available evidence suggests that multi-antibiotic resistant strainsare as easily killed by PDT as naive strains, and that bacteria may notreadily develop resistance to PDT. This may be vital for treating woundsin patients undergoing cancer therapy, HIV patients who demonstrateresistance to antibiotics and the elderly with persistent oralinfections [Hamblin et al. (2004) Photochem Photobiol Sci. 3:436-50].

The device may be used to detect bacteria or micro-organisms in thewound and surrounding normal tissues using low powerexcitation/illumination blue/violet light, but may also be usedimmediately afterwards for destroying them, for example using PDT orother therapies. By using high-power red excitation/illumination light,endogenous porphyrins in bacteria or microorganisms can be destroyedwithin the wound site by PDT. Therefore, this device may have thecapability to serve as an all-in-one non-invasive or non-contact ‘findand treat’ instrument for clinical wound care. Furthermore, oncebacteria or microorganisms are detected, the device may be used to treatand/or disinfect the wound site with PDT, and then the site may bere-imaged soon afterwards to determine the effectiveness of the PDTtreatment. In some embodiments, the device may be used only fordetection/diagnostic purposes only and may not perform any therapeutictreatment itself. The device may be used continuously until the entirewound and surrounding normal tissue have been disinfected, and the woundmay be monitored thereafter in a longitudinal manner as part of standardclinical follow up. Fluorescence images from the device may be used todetermine the biodistribution of the PDT photosensitizer orphotoproducts [Gudgin et al. (1995) J. Photochem. Photobiol. B: Biol.29, 91-93; Konig et al. (1993) J. Photochem. Photobiol. B: Biol. 18,287-290], since most of these are intrinsically fluorescent, and thusthe device may serve as a means to target the PDT treatment light. Thedevice may therefore guide, via imaging, the completeness of the PDTtreatment. Similarly, the device may be used to guide other therapies.

Since some photosensitizers are known to photobleach [Jongen et al.(1997) Phys. Med. Biol. 42, 1701-1716; Georgakoudi et al. (1997)Photochem. Photobiol. 65, 135-144; Rhodes et al. (1997) J. Investig.Dermatol. 108, 87-91; Grossweiner (1986) Lasers Surg. Med. 6, 462-466;Robinson et al. (1998) Photochem. Photobiol. 67. 140-149; Rotomskis etal. (1996) J. Photochem. Photobiol. B: Biol. 33, 61-67] the fluorescenceimaging capability of the device may be used to determine the extent orrate of photobleaching of the photosensitizer. This information may beuseful for optimizing PDT dosimetry [Grossweiner (1997) J. Photochem.Photobiol. B: Biol. 38, 258-268] in order to ensure adequate treatmentof the disease, while at the same time minimizing damage to surroundingnormal tissues. The device, with excitation light sources which may beselected for specific excitation wavelengths and intensities, in anembodiment, may be used to also deliver the light for PDT combined withany commercially available and/or experimental PDT photosensitizers.Therefore, it may have utility in existing clinical PDT indications(e.g., for the skin surface or hollow organs) and/or within the arena ofcommercial/academic research and development of future PDTphotosensitive agents, both pre-clinically and clinically.

FIG. 10G shows an example of the use of the device for monitoring theresponse of bacteria to photodynamic therapy (PDT). Ex vivo porcinetissues were prepared in Petri dishes and contaminated withbioluminescent (BL) Staphylococcus aureus 24 h prior to BL andfluorescence imaging of samples using the device. Bioluminescent andcorresponding fluorescence imaging was performed on a), d)non-contaminated, and b), e) SA-contaminated muscle tissues pre- andpost PDT. Note, Staphylococcus aureus produced red fluorescence color(white arrow in e). PDT was performed on the bacterially-contaminatedmeat sample (marked by a yellow circle) by incubating the sample with acommon photosensitizer called methylene blue (MB) for about 30 mins,followed by removal of excess MB (and rinsing with PBS) and subsequentexposure to about 670 nm light source (here an LED array) for about 10mins at ˜10 J/cm² in order to cause the photodynamic treatment.Comparing the BL intensity scales in b) and c) shows a marked decreasein BL intensity in the treated meat sample following PDT (e.g., PDT haskilled a measureable proportion of the bioluminescent bacteria, thusdecreasing the BL signal intensity), and changes in the fluorescencecharacteristics (e.g., intensity and biodistribution) of theStaphylococcus aureus bacteria (red color) can be seen using thehandheld imaging device following PDT. Note that the intense greenfluorescence on the meat sample (pink arrow in e) was caused byunintentional cross-contamination of the meat sample by non-BLPseudomonas aeruginosa during the experiment (confirmed bybacteriology), and the device detected this. These data suggest the useof the device for monitoring the use of PDT for treatment of bacterialcontamination in biological (and non-biological) samples. (405 nmexcitation; 490-550 nm and >600 nm emission).

This device may be used as an imaging and/or monitoring device inclinical microbiology laboratories. For example, the device may be usedfor quantitative imaging of bacterial colonies and quantifying colonygrowth in common microbiology assays. Fluorescence imaging of bacterialcolonies may be used to determine growth kinetics.

Imaging of Blood in Wounds

Angiogenesis, the growth of new blood vessels, is an important naturalprocess required for healing wounds and for restoring blood flow totissues after injury or insult. Angiogenesis therapies, which aredesigned to “turn on” new capillary growth, are revolutionizing medicineby providing a unified approach for treating crippling andlife-threatening conditions. Angiogenesis is a physiological processrequired for wound healing. Immediately following injury, angiogenesisis initiated by multiple molecular signals, including hemostaticfactors, inflammation, cytokine growth factors, and cell-matrixinteractions. New capillaries proliferate via a cascade of biologicalevents to form granulation tissue in the wound bed. This process may besustained until the terminal stages of healing, when angiogenesis ishalted by diminished levels of growth factors, resolution ofinflammation, stabilized tissue matrix, and endogenous inhibitors ofangiogenesis. Defects in the angiogenesis pathway impair granulation anddelay healing, and these are evident in chronic wounds [Tonnesen et al.(2000) J Investig Dermatol Symp Proc. 5(1):40-6]. By illuminating thetissue surface with selected narrow wavelength bands (e.g., blue, greenand red components) of light or detecting the reflectance of white lightwithin several narrow bandwidths of the visible spectrum (e.g., selectedwavelengths of peak absorption from the blood absorption spectrum ofwhite light), the device may also be used to image the presence of bloodand microvascular networks within and around the wound, including thesurrounding normal tissue, thus also revealing areas of erythema andinflammation.

Reference is now made to FIG. 11. The device may use individual opticalfilters (e.g., 405 nm, 546 nm, 600 nm, +/−25 nm each) in order todemonstrate the possibility of imaging blood and microvasculature inwounds. White light images of a wound may be collected with the deviceand then the device, equipped with a triple band-pass filter (e.g., 405nm, 546 nm, 600 nm, +/−25 nm each), placed in front of the imagingdetector may image the separate narrow bandwidths of blue (B), green(G), and red (R) reflected light components from the wound. Thesewavelength bands may be selected based on the peak absorptionwavelengths of blood, containing both oxygenated and deoxygenatedhemoglobin, in the visible light wavelength range. The resulting imagesmay yield the relative absorption, and thus reflectance, of visiblelight by blood in the field of view. The resulting ‘blood absorption’image yields a high contrast image of the presence of blood and/ormicrovascular networks in the wound and surrounding normal tissues. Theclinician may select the appropriate optical filter set for use with thedevice to obtain images of blood and/or microvascular distributionwithin the wound and the combine this information with one or both ofautofluorescence imaging and imaging with exogenous contrast agents.This may provide a comprehensive information set of the wound andsurrounding normal tissues at the morphological, topographical,anatomical, physiological, biological and molecular levels, whichcurrently may not be possible within conventional wound care practice.

FIG. 11 shows examples of the device used for imaging of blood andmicrovasculature in wounds. The device was used to image a piece offilter paper stained with blood (a) and the ear of a mouse duringsurgery (b). White light images were collected of each specimen usingthe imaging device, in non-fluorescence mode, and then the device wasequipped with a triple band-pass filter placed in front of the imagingdetector (405 nm, 546 nm, 600 nm, +/−25 nm each) to image the separatenarrow bandwidths of blue (B), green (G), and red (R) reflected lightcomponents from the specimens. These wavelength bands were selectedbased on the peak absorption wavelengths of blood in the visible lightwavelength range (inset in a) shows the absorption spectral profile foroxy- and deoxygenated hemoglobin in blood. This shows that using asimple multiband transmission filter, it is possible to combine thethree B, G, R images into a single ‘white light equivalent’ image thatmeasures the relative absorption of light by blood in the field of view.The resulting ‘blood absorption’ image yields a high contrast image ofthe presence of blood containing both oxy- and deoxygenated hemoglobin.The device may be used with narrower bandwidth filters to yield highercontrast images of blood absorption in wounds, for example.

The regulation of angiogenesis over time during wound repair in vivo hasbeen largely unexplored, due to difficulties in observing events withinblood vessels. Although initial tests of the imaging device wereexploratory, simple modification of the existing prototype device mayallow longitudinal imaging of dynamic changes in blood supply andmicrovascular networks during the wound healing process in vivo.

Imaging of Skin and Oral Cavity

This device may be suitable for imaging the skin, the mouth and the oralcavity. The device may allow for detection of connective tissue changesdue to minor cutaneous injuries (e.g., cuts, abrasions) and endogenousbacteria found commonly on normal skin (e.g., Propionibacterium acnes,or P. acnes).

This device may also be suitable for multi-spectral imaging and/ormonitoring of dental plaques, carries and/or cancers in the oral cavity.The device may be used to detect the presence of plaques, periodontaldiseases, carries and cancers, as well as local oral infections, basedon the presence of unique autofluorescence signatures in abnormal orcancerous tissues. The device may use white light, fluorescence, with orwithout autofluorescence or exogenous fluorescent agents, andreflectance imaging to provide real-time detection and diagnosis ofperiodontal disease, plaques, and carries and cancers in the oralcavity. The device may record the images for medical record cataloguing.Unlike the direct (i.e., naked eye) viewing approach used by an existingproduct such as the VELscope System, by Vancouver-based company LEDMedical Diagnostics Inc. (LED-MD), the present device may providedigital imaging and recording of tissue white light, fluorescence andreflectance information.

In dermatology, the device may be used to detect bacteria on normalskin. For example, FIG. 12 demonstrates the high-resolutionautofluorescence imaging of the normal skin of patients faces in whichdistinct red fluorescence from the common bacterium Propionibacteriumacnes is detected.

FIG. 12 shows examples of the use of the device for non-invasivehigh-resolution digital still or video imaging of the oral cavity andthe skin surface in patients. As shown in a), the device may be used forimaging of the mouth and oral cavity. Violet/blue light excitationexcites autofluorescence from the teeth, which appear as an intensegreen fluorescence, compared to the blood rich gums. Periodontal diseaseand caries may be easily detected based on the autofluorescence of theteeth and gum tissues using this device. Red fluorescence at the edge ofthe lips is detected from Propionibacterium acnes (P. acnes) commonlyfound within skin pores. The red fluorescence is produced by endogenousbacterial porphyrins. Note the detection of P. acnes in individual pores(red arrow) on the lip. Similarly, in b), red fluorescence fromendogenous porphyrins in the normal bacteria fauna of the tongue iseasily detected as a bright red fluorescent ‘blanket’ on the tonguesurface. The device may also be used to detect early cancers in the oralcavity based on differences in optical properties (e.g., absorption,scattering, autofluorescence) between normal and pre- and neoplastictissues. The device may be used to ‘scan’ the oral cavity of mucosalcancers, or determine the effects of anticancer therapeutics such asPDT, or other techniques. The device may also be used to image the skinsurface. In c)-e), the device images the skin on patients' faces bydetecting autofluorescence produced by violet/blue light excitation ofthe skin surface. Red fluorescence from P. acnes may easily be detectedin regions of the face (e). The device may be used to image and/ormonitor the potential effects of dermatological interventions (e.g.,topical creams, drugs and other antibiotics, etc.) on patients' skin. Inf) and g), the device was also used to image minor cuts (arrow, h),scrapes and abrasions on patients' skin, as well as psoriasis on afinger (arrow, i). Under violet/blue light, the device detected tissueautofluorescence from connective tissue components (e.g., collagen andelastin) from the wound site and surrounding normal skin to yieldhigh-resolution images of subtle cutaneous lesions. P. acnes is thecausative agent of acne vulgaris (i.e., pimples) and is a commonresident of the pilosebaceous glands of the human skin, and is occultunder white light visualization. These autofluorescent images wereobtained without the need of exogenous agents/drugs and demonstrate thecapability of the device to detect bacteria porphyrin fluorescence insingle skin pores.

FIG. 12J shows an example of the use of the imaging device for real-timefluorescence detection of common bacterial flora on skin. a) Redfluorescence on and around the nose is detected from Propionibacteriumacnes (P. acnes) commonly found within skin pores. b) Fluorescenceimaging may also be used to detect and monitor more than one bacterialspecies on the skin at the same time, for example Propionibacteriumacnes appear as red fluorescent (red arrow) while Pseudomonas aeruginosaappear bright green (green arrows). These data suggest the use of thedevice for distinguishing relative concentrations/levels of variousbacterial species, determining their biodistributions on body surface,and monitoring response to anti-bacterial treatments in dermatology andcosmetology applications. c) Shows an example of a fluorescence image ofa culture grown on agar from a swab taken from normal skin on the noseof a healthy volunteer. Bacteriology results showed the presence ofPseudomonas aeruginosa.

Such a capability to image and document the presence and biodistributionof bacteria on the skin surface makes the device potentially useful inthe dermatology and cosmetology fields. For example, fluorescenceimaging may be performed prior to, during and after application ofdermatological treatment and/or pharmaceutical/cosmetic formulations(e.g., topical creams, drugs and other antibiotics, skin disinfectingagents, acne treatments, etc.) to the normal and abnormal skinconditions, including but not limited to scarring, hyper-pigmentation,acne, psoriasis, eczema, rashes, etc. Fluorescence/reflectanceimage-guided tattoo removal (e.g., using surgery or available lasertreatments) may also be an option with the device. The device was alsoused to image minor cuts, scrapes and abrasions on patients skin andunder violet/blue light, tissue autofluorescence from connective tissuecomponents (e.g., collagen and elastin) from the wound site andsurrounding normal skin aided in detecting white light-occult changes inconnective tissues during minor cutaneous wound healing (as seen in FIG.12 h, i). In addition, the device may also serve as a practical,cost-effective and sensitive image-based tool for early detection ofoccult skin cancers and non-cancerous (i.e., benign) lesions in anon-invasive manner [Chwirot et al. (1998) Eur J Cancer. 34(11):1730-4].The device may then be used to provide image-guidance for surgicalexcision of the lesions or for PDT. For the latter, fluorescence imagingmay monitor PDT response and determine completeness of treatmentover-time with multiple longitudinal image scans of the affected area.The device may be used in real-time for determining PDT photosensitizerlocalization and biodistribution and photobleaching, and this may bemapped onto the white light image of the area to be treated foranatomical comparison. Changes in the optical properties between normaland diseases or burned tissues may be detected using both then whitelight and fluorescence imaging capabilities of the device. The devicemay also be used to image, assess and longitudinally monitor the healingprocess in burns or the determine response of skin grafts or temporaryskin substitutes in treatment of burn patients [Bishop (2004) Crit CareNurs Clin North Am. 200416(1):145-77]. The device may also serve todetect and monitor late radiation-induced skin damage during treatmentof patients with ionizing radiation [Charles (2007) J Radiol Prot.27(3):253-74].

In addition, the device may be used to image the mouth and oral cavity,particularly in the embodiment where the device is small and compact.Pilot imaging studies showed that the device may detect endogenousbacteria in the oral cavity (e.g., on the tongue surface and betweenteeth on the gum line), suggesting a use in clinical detection of cariesand periodontal disease [Pretty (2006) J Dent. 34(10):727-39].Additionally, tissue autofluorescence has been shown to be useful indetecting oral cancers [Kois et al. (2006) Dent Today. 25(10):94, 96-7].The device may be used to detect early cancers in the oral cavity basedon differences in optical properties (e.g., absorption, scattering,autofluorescence) between normal, pre- and neoplastic oral tissues. Inaddition, the device may be used to ‘scan’ the oral cavity for mucosalcancers, and monitor the response to therapy.

In general, the device may be used to image and/or monitor targets suchas a skin target, an oral target, an ear-nose-throat target, an oculartarget, a genital target, an anal target, and any other suitable targetson a subject.

Use in Malignant Wounds

A malignant wound is also known as tumor necrosis, a fungating wound,ulcerating cancerous wound, or malignant cutaneous wound. A malignantwound can be an emotional and physical challenge for patients, familiesand even for the experienced clinician. Fungating and ulcerating woundscan be unsightly, malodorous and painful. These wounds may be indicatorsof disease progression, and may become infected leading todelayed/impeded healing and associated morbidity and thus, reducedquality of life for patients.

Many cancer patients live with the knowledge that their disease is bothprogressive and incurable. For a significant minority of these peoplethis reality may be present in the form of a malodorous, exuding,necrotic skin lesion, which can be a constant physical reminder ofdisease progression (Mortimer P S. In: Doyle et al. editors. OxfordTextbook of Palliative Medicine (2nd ed). Oxford: Oxford UniversityPress, 1998, 617-27; Englund F. RCN Contact 1993; Winter: 2-3). Theselesions are commonly known as ‘fungating wounds’, the term ‘fungating’referring to a malignant process of both ulcerating and proliferativegrowth (Grocott P. J Wound Care 1995; 4(5): 240-2). Lesions that have apredominantly proliferative growth pattern may develop into a nodular‘fungus’ or ‘cauliflower’ shaped lesion, whereas a lesion that isulcerating will produce a wound with a crater-like appearance (GrocottP. J Wound Care 1999, 8(5): 232-4; Collier M. Nurs Times 1997; 93(44):suppl 1-4). Such lesions may also present with a mixed appearance ofboth proliferating and ulcerating areas (Young T. Community Nurse 1997;3(9): 41-4).

A malignant wound may develop in one of the following ways:

-   -   As a result of a primary skin tumour such as squamous cell        carcinoma or melanoma.    -   Through direct invasion of the structures of the skin by an        underlying tumour, for example breast cancer, or haematological        malignancy such as cutaneous T-cell lymphoma (mycosis        fungoides).    -   From metastatic spread of a distant tumour. Metastasis may occur        along tissue planes, capillaries or lymph vessels.

Malignant wounds are often difficult to manage related to theirlocation, odor, excessive exudates, and propensity for bleeding. Everymalignant wound may be unique in its appearance and presenting symptoms.The common symptoms associated with malignant wounds include malodor,excessive exudates, infection, bleeding, maceration and excoriation ofperi wound skin, pruritis, pain, poor aesthetics and cosmetic effects ofdressings. Currently, the approach to care is mainly holistic andprimarily palliative with the aim to control symptoms at the wound siteand reduce the impact of the wound on the patient's daily life,primarily by identifying bacterial/microbial infection(s) and monitoringfor signs of healing. Unless the pathology is controlled these woundsare not expected to heal.

The described device may be useful for performing clinical assessment ofsuch wounds (e.g., physical and biological examination). The device mayprovide: a means of thorough image-based wound assessment at baselineand at regular intervals throughout treatment (i.e., longitudinalmonitoring), wound assessment including location, size of wound, color,type and amount of any discharge or drainage, serial white light (e.g.,for color changes) and fluorescence (e.g., for tissue structural,cellular, biological, and molecular changes) images of chronic malignantwounds, and may provide assessment of any signs and symptoms ofinfection in real-time, that would affect treatment planning andefficacy. The device may be integrated into the current clinicalpractice for assessment and care of such malignant wounds.

Imaging of Exogenous Fluorescence Contrast Agents

The development of highly efficient analytical methods capable ofprobing biological systems at system level is an important task that isrequired in order to meet the requirements of the emerging field ofsystems biology. Optical molecular imaging is a very powerful tool forstudying the temporal and spatial dynamics of specific biomolecules andtheir interactions in real time in vivo. Several recent advances inoptical molecular imaging have occurred, such as the development ofmolecular probes that make imaging brighter, more stable and morebiologically informative (e.g., FPs and semiconductor nanocrystals, alsoreferred to as quantum dots), the development of imaging approaches thatprovide higher resolution and greater tissue penetration, andapplications for measuring biological events from molecule to organismlevel. These advances may also be applied to disease diagnosis (e.g.,wound care) and pharmaceutical screening. However, current fluorescenceimaging devices are large, complicated and involve expensive opticalcomponents and very sensitive camera detectors which makes such systemsextremely expensive. The device developed here offers an alternative tothese cost-limiting systems for preclinical or research studies as wellas possible clinical translation of such methods.

Reference is now made to FIG. 13. The device was also used to image theanimal for general observation under fluorescence to determine theextent of fluorescence from the BPD photosensitizer throughout the skinsurface. FIG. 13 demonstrates utility of the device in for real-timeimaging and sensitive detection of exogenous fluorescence contrastagents in vivo (e.g., quantum dots, QDots). In a), the device was usedto image exogenous fluorescence contrast agents in a sacrificed ratbearing human breast tumor cells metastasized to the bone in the hindleg. The rat was previously injected with a fluorescence photosensitizercalled benzo-porphyrin derivative (BPD) for an unrelated photodynamictherapy experiment. The rat was administered two separate fluorescentsemiconductor nanoparticle solutions (here, QDots), each emittingfluorescence at 540 (+/−15) nm and 600 (+/−15) nm solutions viasubcutaneous injection in the left hind leg. Injections wereapproximately 1 cm apart. The device was then used to image the wholebody of the rat using violet/blue excitation light. The rate skinappeared red, and this was likely due to the combination of thefluorescence from the benzo-porphyrin derivative (BPD) photosensitizeradministered to the rat prior to the experiment, which was forsubsequent PDT, as well as dust and food contamination from the cage inwhich rat was housed.

Referring still to FIG. 13, in b) the fluorescence from the green andred QDots (inset) was easily detected beneath the skin at the site ofthe injection, with the red QDots emitting the brighter signal, due togreater tissue penetration of red light. c) shows a magnified image ofthe hind leg shown in b). The device was capable of detecting multiplefluorescence contrast agent simultaneously along with background tissueautofluorescence with sufficient signal-to-noise (green and red arrows)so as to permit its use in preclinical and expected clinicalfluorescence imaging of multiplexed molecularly-targeted fluorescencecontrast agents in vivo. Note the green fluorescence is weaker than thered because both the violet/blue excitation light and the subsequentgreen QDot fluorescence are preferentially absorbed by blood and redQDot fluorescence light has a greater penetration depth through tissue.In d), the device was also used to image the animal for generalobservation under fluorescence to determine the extent of fluorescencefrom the BPD photosensitizer throughout the skin surface. The device mayalso be useful for guiding intravenous injections using needles bydetecting surface blood vessels beneath the skin. The device may thus beused to detect fluorescent tumors, such as those that are transfectedwith fluorescent proteins and grown subcutaneously in a xenograft ororthotopic model. Thus, the device may be used for visualizing multiplewound healing and/or infectious biomarkers using multiplexed exogenousfluorescent molecular targeting agents (e.g., for in situ image-basedbacteriology).

To improve the use of fluorescence contrast agents in preclinicalresearch and eventually for clinical translation of optical molecularimaging technologies, it is desirable to be able to relatively rapidlydifferentiate and identify various fluorescent agents. In e) and f), thedevice was also used as a means of relatively rapidly identifying whichfluorescence contrast agents were in the syringes prior to injection,which was not possible under standard white light, demonstrating theutility of the device as a cost-effective fluorescence-image guidedtechnology for providing useful information quickly duringfluorescence-image guided surgical and/or PDT procedures, wherefluorescent compounds are commonly used, possibly even in emerging woundcare techniques.

Fluorescence-Image Guided Surgery

An emerging area is the use of fluorescence imaging for diagnosticscreening and image-guided surgery. Overcoming limitations of standardsurgery using white light, fluorescence images may be used to aid insurgical resection of tumors in vivo based on fluorescence (e.g., eitherautofluorescence or fluorescence from exogenous targeted/non-targetedcontrast agents) as well as checking for completeness of tumor removal(e.g., clear margins). Fluorescence-image guided surgery hasdemonstrated improvements in survival, pre-clinically and clinically[Bogaards et al. (2004) Lasers Surg Med. 35:181-90]. For example, duringexploratory surgery on a rat, the device may provide standard whitelight imaging of the surgical field.

Reference is now made to FIG. 14. Several tests were conducted todemonstrate the utility of the device for fluorescence-image guidedsurgery in small animals. Exploratory surgery was performed on aeuthanized female rat using the imaging device. FIG. 14 shows examplesof the use of the device for fluorescence-image guided surgery usingimaging contrast agents. During exploratory surgery, the device providedstandard white light imaging of the surgical field, here, the abdomen ofa female rat (a). The surgeon used the viewing screen of the device toguide the procedure, switching easily and rapidly between white lightand fluorescence mode. In b), using violet/blue excitation light, thedevice provided added contrast between different types of tissues, whichwas not possible during white light imaging. For example, connectivetissues in the appeared bright green fluorescent (green arrow), whilethe skin surface (with the red fluorescent photosensitizer BPD) appearedred (red arrow), and the QDots previously injected into the hind legappeared a bright red (blue arrow). Fluorescence imaging was used todetect contamination of surgical instruments and equipment (e.g., gauze,tape, blankets, etc.) during the surgical procedure. In c), the devicealso demonstrated utility by detecting soiled/contaminated surgicalgauze during the procedure. Compared with standard white light underwhich all gauze appeared clean, the gauze used to clean the skin and thesurgical field during surgery appeared red fluorescent (left) comparedwith clean gauze (right).

The device was also used for real-time detection of exogenousfluorescent contrast agents (e.g., for labeled cell tracking and fate invivo experiments, for tissue engineering studies in regenerativemedicine, etc.) in an animal model. For this, during surgery, the devicewas used in fluorescence mode to image the presence of red fluorescentQDots injected within the heart muscle and lungs of the rat (d). Underviolet/blue excitation light, the red QDots can be easily detectedwithin the heart (e) and the lungs (f), which appear dark due to thehigh concentration of blood in these organs, demonstrating the utilityof the device for guiding and targeting biopsies or microsurgicalprocedures, especially those aimed at detection and removal of cancers(e.g., using autofluorescence or fluorescence contrast enhancement).Note the bright red autofluorescence detected by the device fromdigested food material in the colon. In g), the device demonstrated itsutility in imaging fluorescent tumor phantoms commonly used in smallanimal imaging research. Solid spherical polymer tumor phantoms dopedwith fluorescent dye were prepared in varying sizes and placed withinthe surgical field to demonstrate the capability of the device inproviding rapid ‘high contrast’ fluorescence imaging in small animalcancer models.

These results show that the device may be useful in detecting sub-mmsized lesions with fluorescence guidance, which may be useful fortargeting biopsies or microsurgical procedures, especially those aimedat detection and removal of cancers (e.g., using autofluorescence orfluorescence contrast enhancement). The device also may have utility inimaging fluorescent tumor phantoms commonly used in small animal imagingresearch.

FIG. 15 shows examples of the device being used for video recording ofhigh-resolution fluorescence-image guided surgery of the rat in FIG. 9.The device may be capable of providing both still digital images andmovies taken with standard white light (WL) (a) and fluorescence (FL)(b), which may be switched between easily. Here, the device was used tocapture digital movies of a surgical procedure on a rat using both whitelight and fluorescence imaging. The surgeon used the digital displayscreen of the device to guide the complete surgical procedure usingfluorescence where white light failed to provide adequate information.In c)-e), for example, under violet/blue light excitation, fluorescenceimaging provided the surgeon with significant image contrast betweendifferent types of tissues. Blood vessels can be seen clearly underfluorescence, and connective tissues can be discerned from thegastrointestinal tract. Digested food material can also bedistinguished. The device may provide a real-time imaging solution forimage-guided surgical intervention or biopsy allowing the surgeon tomake critical judgments during the procedure. Digital still and/or moviecapture of the surgery may allow retrospective analysis of the procedurefor patient health records and future skills training of medicalpersonnel. The device may also record audio during the surgicalprocedure thus allowing a complete record to be collected of eachprocedure. The utility of the device was also demonstrated as a highlyuseful tool for image-guided minimally-invasive micro-surgery inanimals, and potentially in human procedures.

FIG. 16 shows examples of the device being used forautofluorescence-image guided surgical resections of tissues in a mousecardiac infarction model (a). During exploratory surgery, the deviceprovided standard white light (WL) imaging of the open surgical field,here, the abdomen of the mouse (b). The surgeon used the viewing screenof the device to guide the procedure, switching easily and rapidlybetween white light and fluorescence mode. Using violet/blue excitationlight, the device provided high-contrast between different types oftissues, which was not possible during white light imaging (c). Forexample, various internal organs were visualized using high-resolutionautofluorescence imaging. In d), the intact animal can be imaged withfluorescence prior to and during surgery (e).

FIG. 17 shows examples of the device being used for non-invasivereal-time autofluorescence-image guided surgery of a mouse brain. Duringexploratory surgery, the device provided standard white light (WL)imaging of the open surgical field (a), here, the skull of the mouse canbe seen. The surgeon used the viewing screen of the device to guide thesurgical procedure, switching easily and rapidly between WL andfluorescence (FL) mode. b) shows the view of the surgical field (here,skull intact) provided by the imaging device under tissueautofluorescence. Note the surgical area is dark, mainly due toabsorption of the violet/blue excitation light and the resultingautofluorescence caused by blood. The snout and eyes appear bright redfluorescent compared to the bright green fluorescence from the fur. c)shows the surgical field with the skull cap removed under WL, while d)shows the autofluorescence image of the brain surface using the imagingdevice with violet/blue excitation light. Injection of an exogenouscontrast agent (here, red fluorescent quantum dots) directly into theright hemisphere of the brain produces a bright red fluorescence(arrows) (e). This demonstrates the utility of the device for imagingfluorescence contrast agents, specifically for high-resolutionfluorescence-image guided surgery.

Use in Clinical Care

Although current wound management practice aims to decrease themorbidity and mortality of wounds in patients, a limitation is theavailability of health care resources. The potential of incorporatingthe technology of telemedicine into wound care needs is currently beingexplored. Wound care is a representation of the care of chronic anddebilitating conditions that require long-term specialized care. Themajor effect of improved living conditions and advances in health careglobally has led to people living longer. Therefore, the percentage ofworlds' elderly and those with chronic medical conditions that wouldrequire medical attention is rising. With the escalating costs of healthcare, and the push of the industry towards outpatient care, this is apart of the health care crisis that is demanding immediate attention.

The present device may provide biologically-relevant information aboutwounds and may exploit the emerging telemedicine (e.g., E-health)infrastructure to provide a solution for mobile wound care technologyand may greatly impact wound health care treatment. Wound care accountsfor a large percentage of home visits conducted by nurses and healthcare workers. Despite best practices some wounds do not heal as expectedand require the services of a clinical specialist. The device describedhere may enable access to specialized clinical resources to help treatwounds from the convenience of the patient's home or chronic carefacility, which decreases travel time for clients, increasesavailability to clinical wound specialists, and may reduce costs to thehealth care system.

Different uses of the imaging device have been discussed for woundassessment, monitoring and care management. The device may be used todetect and monitor changes in connective tissues (e.g., collagen,elastin) and blood/vascular supply during the wound healing process,monitor tissue necrosis and exudate in wounds based on fluorescence,detect and diagnose wound infections including potentially indicatingcritical ‘clinically significant’ categories of the presence of bacteriaor micro-organisms (e.g., for detecting contamination, colonization,critical colonization and infection) at the surface and deep withinwounds [Kingsley, Ostomy Wound Manage. 2003 July; 49(7A Suppl):1-7],provide topographic information of the wound, and identify wound marginsand surrounding normal tissues. Tissue fluorescence and reflectanceimaging data may be ‘mapped’ onto the white light images of the woundthereby permitting visualization within the wound and the surroundingnormal tissues of essential wound biochemical and photobiological (e.g.,fluorescence) information, which has not been possible to date.Real-time imaging of wounds may be performed over time to monitoringchanges in wound healing, and to potentially monitor the effectivenessof treatments by providing useful information about underlyingbiological changes that are occurring at the tissue/cellular level(e.g., matrix remodeling, inflammation, infection and necrosis). Thismay provide quantitative and objective wound information for detection,diagnosis and treatment monitoring in patients. In particular, thedevice may be used to monitor and/or track the effectiveness of therapyat a biological level (e.g., on a bacterial level), which may providemore information than monitoring only the macroscopic/morphologicalappearance using white light.

The device may provide real-time non-invasive image-guided biopsytargeting, clinical procedural guidance, tissue characterization, andmay enable image-guided treatment using conventional and emergingmodalities (e.g., PDT). In addition, use of the imaging device may beused to correlate critical biological and molecular wound informationobtained by fluorescence (e.g., endogenous tissue autofluorescenceand/or administration of exogenous molecular-biomarker targetedfluorescence contrast agents) with existing and emerging clinical woundcare assessment and treatment guides, such as the NERDS and STONESguidelines proposed by Sibbald et al. (Sibbald et al. IncreasedBacterial Burden and Infection: The Story of NERDS and STONES. ADV SKINWOUND CARE 2006; 19:447-61). The fluorescence imaging data obtained withthe device may be used to characterize, spatially and spectrally,bacterial balance and burden at the superficial and deep levels ofwounds. The device may provide real-time non-invasive image-guidedbiopsy targeting, clinical procedural guidance, tissue characterization,and may enable image-guided treatment using conventional and emergingmodalities (e.g., photodynamic therapy, PDT). The device may be usedwithin the clinical setting and integrated into conventional clinicalwound care regimens, and may have a distinct role in areas of infectiousdiseases. It should be noted as well that this device may also be usedfor real-time analysis, monitoring and care for chronic and acute woundsin animals and pets, via conventional veterinary care.

This device may allow real-time wound healing assessment for a largepatient cohort base. In particular, elderly people, diabetics,immuno-suppressed and immobilized individuals have an increasedincidence of chronic wounds and other dermal afflictions that resultfrom poor circulation and immobility, e.g. pressure ulcers such as bedsores, venous stasis ulcers, and diabetic ulcers. These chronicconditions greatly increase the cost of care and reduce the patient'squality of life. As these groups are growing in number, the need foradvanced wound care products will increase. This device may impactpatient care by allowing a cost-effective means of monitoring chronicand acute wounds in a number of settings, including hospitals,ambulatory clinics, chronic care facilities, in-home-visit health care,emergency rooms and other critical areas in health care facilities.Further, such a ‘hand-held’ and portable imaging device may be easilycarried and used by nursing and ambulance staff. Early identification ofscarring, which is related to connective tissue production andre-modeling of the wound, and bacterial infections may be detected andtreated appropriately, something that is currently difficult. Inaddition, recent developments in advanced wound-care products includingmultiple dressing types (e.g., film, hydrocolloid, foam, anti-microbial,alginate, non-adherent, impregnated), hydrogels, wound cleansers anddebriding agents, tissue engineered products (e.g., skin replacements,substitutes, and tissue-engineered products such as syntheticpolymer-based biological tissue and growth factors), wound cleansers,pharmacological products, and physical therapies may also benefit fromthe device developed here as it may allow image-based longitudinalmonitoring of the effectiveness of such treatments. Physical therapiesmay include hydrotherapy, electrical stimulation, electromagneticstimulation devices, ultraviolet therapy, hyperbaric oxygen therapy,ultrasound devices, laser/light emitting diode (LED) devices, and woundimaging/documentation.

Wound tissue analysis is typically required for the assessment of thehealing of skin wounds. Percentage of the granulation tissue, fibrin andnecrosis in the wound, and their change during treatment may provideuseful information that may guide wound treatment. Image analysis mayinclude advanced statistical pattern recognition and classificationalgorithms to identify individual pixels within the fluorescence woundimages collected with the device based on the optical information of thewound and surrounding normal tissue. Thus, image analysis may allowwound images to be mapped into various components of the wound,including total wound area, epithelialization, granulation, slough,necrotic, hypergranulation, infected, undermining, and surroundingtissue margins. This has an added advantage of providing relativelyrapid determination of wound healing rates, as well as informing guidepatient management decisions.

FIG. 25 illustrates the projected management workflow for the imagingdevice in a clinical wound care setting. The device may be easilyintegrated into routine wound assessment, diagnosis, treatment andlongitudinal monitoring of response, and may provide critical biologicaland molecular information of the wound in real-time for rapiddecision-making during adaptive interventions.

This device may be easily integrated into existing health-care computerinfrastructures (e.g., desktop and pocket PCs used by a growing numberof physicians or other health care professionals) for longitudinal imagecataloguing for patient wound management within the conventionalclinical environment. The wireless receiving and transmission of datacapabilities of the device may allow monitoring of wound care andhealing remotely through existing and future wireless telemedicineinfrastructure. The device may be used to transfer essential medicaldata (e.g., wound health status) via the internet or over wirelessservices, such as cellular telephone, PDA or Smartphone services, toremote sites which may permit remote medical interventions, with afurther utility in military medical applications for battlefield woundmanagement. The device may allow real-time surface imaging of woundsites and may be easily carried by point-of-care personnel in clinicalsettings. Using cost-effective highly sensitive commercially availabledigital imaging devices, such as digital cameras, cellular phones, PDAs,laptop computers, tablet PCs, webcams, and Smart phones, etc. as theimage capture or recording component, the device may offer image-baseddocumentation of wound healing and tracking of treatment effectiveness.Also, this technology may be adapted to also function in ‘wireless’ modeto permit remote medical interventions by potentially adapting it foruse with high-resolution digital cameras embedded incommercially-available cellular telephones.

By using web-based telemedicine and remote medical monitoringinfrastructure, the imaging device may be integrated into a‘store-and-forward’ concept of wound assessment systems. In addition toproviding digital images, such a system may present a comprehensive setof clinical data that meet the recommendations of clinical practiceguidelines. The presently-disclosed device may integrate into acomputer-based wound assessment system (e.g., with image analysissoftware) to be used by a health care facility to enhance existingclinical databases and support the implementation of evidence—basedpractice guidelines. Such an integrated telemedicine infrastructure maybe used for monitoring patients at home or in long-term-care facilities,who may benefit from routine monitoring by qualified clinicians butcurrently do not have access to this care. This device may be furtherdeveloped into a portable handheld point-of-care diagnostic system,which may represent a major advance in detecting, monitoring, treating,and preventing infectious disease spread in the developed and developingworlds. This knowledge may significantly improve the diagnostic toolsavailable to practitioners who treat chronic wounds in settings wherequantitative cultures are inaccessible.

The device may allow digital imaging with optical and digital zoomingcapabilities (e.g., those embedded in commonly available digital imagingdevices). Still or video image quality may be in ‘high-definition’format to achieve high spatial resolution imaging of the tissue surface.Images may be recorded as still/freeze frame and/or in video/movieformat and printed using standard imaging printing protocols which do(e.g., connected via USB) or do not (e.g., PictBridge) require apersonal computer. The images/video data may be transferred to apersonal computer for data archival storage and/or image viewing and/oranalysis/manipulation. The device may also transfer data to a printer orpersonal computer using wired or wireless capabilities (e.g.,Bluetooth). Visualization may be performed on the hand-held devicescreen and/or in addition to simultaneous viewing on a videoscreen/monitor (e.g., head-mounted displays and glasses) using standardoutput video cables. This device may display, in combination orseparately, optical wavelength and fluorescence/reflectance intensityinformation with spatial dimensions of the imaged scene to allowquantitative measurements of distances (e.g., monitoring changes tissuemorphology/topography) over time. The device may also allow digitalimage/video storage/cataloguing of images and related patient medicaldata, for example using dedicated software with imaging analysiscapabilities and/or diagnostic algorithms.

Image Analysis

Image analysis may be used together with the device to quantitativelymeasure fluorescence intensities and relative changes in multiplefluorescence spectra (e.g., multiplexed imaging) of the exogenousoptical molecular targeting probes in the wound and surrounding normaltissues. The biodistributions of the fluorescent probes may bedetermined based on the fluorescence images collected and these may bemonitored over time between individual clinical wound imaging sessionsfor change. By determining the presence and relative changes inabundance quantitatively, using the device, of each and all of thespectrally-unique fluorescent probes, the clinical operator maydetermine in real-time or near real-time the health and/or healingstatus and response to treatment over time of a given wound, for exampleby using a look-up table in which specific tissue, cellular andmolecular signals are displayed in correlation to wound health, healingand response status, an example of which is shown in FIG. 21 (adaptedfrom Bauer et al., Vasc & Endovasc Surg 2005, 39:4). This may permit theclinician to determine whether a wound is healing based on biologicaland molecular information which may not be possible otherwise withexisting technologies. Furthermore, the presence and abundance ofbacteria/microorganisms and their response to treatment may offer ameans to adapt the therapy in real-time instead of incurring delays inresponse assessment with conventional bacteriological testing of woundcultures.

Image analysis techniques may be used to calibrate the initial or firstimages of the wound using a portable fluorescent standard placed withinthe field of view during imaging with the device. The image analysis mayalso permit false or pseudo color display on a monitor fordifferentiating different biological (e.g., tissue, cellular, andmolecular) components of the wound and surrounding normal tissuesincluding those biomarkers identified by autofluorescence and thoseidentified by the use of exogenous targeted or untargetedfluorescence/absorption contrast agents.

Examples of such biomarkers are listed in FIG. 22 (adapted from Brem etal. Journal of Clinical Investigation, 117:5, 2007) and illustrated inFIG. 23. In FIG. 23, the diagram shows mechanisms of wound healing inhealthy people versus people with diabetic wounds. In healthyindividuals (left), the acute wound healing process is guided andmaintained through integration of multiple molecular signals (e.g., inthe form of cytokines and chemokines) released by keratinocytes,fibroblasts, endothelial cells, macrophages, and platelets. Duringwound-induced hypoxia, vascular endothelial growth factor (VEGF)released by macrophages, fibroblasts, and epithelial cells induces thephosphorylation and activation of eNOS in the bone marrow, resulting inan increase in NO levels, which triggers the mobilization of bone marrowEPCs to the circulation. For example, the chemokine SDF-1α □ promotesthe homing of these EPCs to the site of injury, where they participatein neovasculogenesis. In a murine model of diabetes (right), eNOSphosphorylation in the bone marrow is impaired, which directly limitsEPC mobilization from the bone marrow into the circulation. SDF-1αexpression is decreased in epithelial cells and myofibroblasts in thediabetic wound, which prevents EPC homing to wounds and therefore limitswound healing. It has been shown that establishing hyperoxia in woundtissue (e.g., via HBO therapy) activated many NOS isoforms, increased NOlevels, and enhanced EPC mobilization to the circulation. However, localadministration of SDF-1α □ was required to trigger homing of these cellsto the wound site. These results suggest that HBO therapy combined withSDF-1α □ administration may be a potential therapeutic option toaccelerate diabetic wound healing alone or in combination with existingclinical protocols.

Pre-assigned color maps may be used to display simultaneously thebiological components of the wound and surrounding normal tissuesincluding connective tissues, blood, microvascularity, bacteria,microorganisms, etc. as well as fluorescently labeleddrugs/pharmacological agents. This may permit visualization in real-timeor near real-time (e.g., less than 1 minute) of the health, healing andinfectious status of the wound area.

The image analysis algorithms may provide one or more of the followingfeatures:

Patient Digital Image Management

-   -   Integration of a variety of image acquisition devices    -   Records all imaging parameters including all exogenous        fluorescence contrast agents    -   Multiple scale and calibrations settings    -   Built-in spectral image un-mixing and calculation algorithms for        quantitative determination of tissue/bacterial autofluorescence        and exogenous agent fluorescence signals    -   Convenient annotation tools    -   Digital archiving    -   Web publishing

Basic Image Processing and Analysis

-   -   Complete suite of image processing and quantitative analysis        functions Image stitching algorithms will allow stitching of a        series of panoramic or partially overlapping images of a wound        into a single image, either in automated or manual mode.    -   Easy to use measurement tools    -   Intuitive set up of processing parameters    -   Convenient manual editor

Report Generation

-   -   Powerful image report generator with professional templates        which may be integrated into existing clinical report        infrastructures, or telemedicine/e-health patient medical data        infrastructures. Reports may be exported to PDF, Word, Excel,        for example.

Large Library of Automated Solutions

-   -   Customized automated solutions for various areas of wound        assessment including quantitative image analysis.

Although image analysis algorithm, techniques, or software have beendescribed, this description also extends to a computing device, asystem, and a method for carrying out this image analysis.

Stem Cell Therapy and Cancer Monitoring

The device may be used for imaging and detection of cancers in humansand/or animals. The device may be used to detect cancers based oninherent differences in the fluorescence characteristics between suchcancers and surrounding normal tissues in patients. This device may alsobe used for image-based detection of cancers in pets, for example withinveterinary settings.

The device may also be used as a research tool for multi-spectralimaging and monitoring of cancers in experimental animal models of humandiseases (e.g., wound or cancers). The device may be used to detectand/or image the presence of cancers and track tumor growth in animalsmodels of cancer, particularly using fluorescent (e.g., in the visibleand NIR wavelength ranges) protein transfected tumor cell lines.

The imaging device may be used in conjunction with both existing andemerging cell therapies useful for reconditioning of chronic wounds andaccelerating their healing. For this, fluorescently labeled stem cellsmay be administered to the wound site prior to imaging with the device.Pluripotential stem cells (PSCs), the precursors to all more specializedstem cells, are capable of differentiating into a variety of cell types,including fibroblasts, endothelial cells and keratinocytes, all of whichare critical cellular components for healing. A recent report on anuncontrolled clinical trial suggests that direct application ofautologous bone marrow and its cultured cells may accelerate the healingof non-healing chronic wounds (Badiavas et al. Arch Dermatol 2003;139(4): 510-16). Considering the pathophysiological abnormalitiespresent in chronic wounds there is the potential that stem cells mayreconstitute dermal, vascular and other components required for optimalhealing. The device may be used to visualize and track the labeled stemcells at the wound site over time, and determine their biodistributionand therapeutic effect. Using exogenous fluorescence molecular-targetedagents, for example as described above, may confirm differentiation ofthe stem cells in vivo and may also aid in determining the response ofthe wound to this treatment.

For example, this device may be used to identify, track and/or monitorcancer tumor stem cells and stem cells in general (e.g., in preclinicalsmall animal experimental models of cancers and other clinical models).An example is shown in the Figures. The device may also be useful forimaging of clinical cell therapies, including treatment of diseasesusing stem cells.

Reference is now made to FIG. 18. In a), a mouse model is shown usingwhite light. In b), the individual organs of the mouse are clearly seenusing the fluorescence imaging device. c) shows the liver of the mouseimaged with the device, and not fluorescence is seen. d) shows the lungsof the mouse in white light. e) shows the lungs of the mouse imaged withthe device, with the cancer tumor stem cells clearly seen as brightfluorescent spots.

Referring now to FIG. 19, in a), the liver of the mouse model of FIG. 18is not visible under fluorescence imaging. b), d) and f) show differentviews of the mouse lungs under white light. c), e) and g) showcorresponding view of the mouse lungs imaged using the device, clearlyshowing cancer tumor stem cells as bright fluorescent spots.

FIG. 19H shows an example of the use of the device for detection ofhuman ovarian tumor-bearing nude mice. a) White light image ofvirus-treated and non-treated control mice, showing open abdominalcavity. b) Corresponding, fluorescence image of treated and control miceshows orange-red fluorescence from the optically-labeled virus in tumornodules in the messentary (yellow arrows), compared with control. c)Shows a magnified view of the messentaries, illustrating thebiodistribution of the virus optical probe within the tumor nodules, aswell as the capability to detect sub-millimeter tumor nodules (bluearrow), compared with d) control mouse. Note, that probe-fluorescencemay be differentiated from background intestinal tissueautofluorescence. These data illustrate the potential use of the devicefor imaging treatment response including, but not limited to, forexamples, virotherapies and cell therapies, as well as for image-guidedsurgical resection of fluorescent tumor samples (c; insets) (405 nmexcitation, 500-550 nm emission (green), >600 nm emission (red)).

FIG. 19I shows an example of the use of the device fordetection/visualization in mouse colon tumor-bearing nude miceadministered a fluorescent cocktail of separate exogenous green and redtumor cell-targeting probes post-operatively. a) White light and b)corresponding multispectral fluorescence image of the open abdominalcavity showing simultaneous detection of both the green (green arrow)and red (red arrow) molecular probes, which may be analyzed withspectral un-mixing software. The device may be modified to permitendoscopic imaging as well. In this example, c) a rigid endoscopic probewas attached to the handheld imaging device and d) white light and e)fluorescence images were obtained of tissue surgically resected from themouse in image a), b). These data suggest the use of the device withendoscopic probe accessories for portable endoscopic real-timefluorescence imaging in vivo in human and veterinary patients for avariety of detection, diagnostic or treatment monitoring applications(clinical- and research-based). f) The device (e.g., with endoscopiccapabilities) may be capable of fluorescence imaging of multiplespectrally-unique “probes” which may be used in vivo (405 nm excitation;490-550 nm and >600 nm emission channels).

This device may be used for multi-spectral imaging and detection ofcancers in humans and animals. This device may be also used to detectcancers based on inherent differences in the fluorescencecharacteristics between such cancers and surrounding normal tissues inpatients. This device may also be used for image-based detection ofcancers in animals such as pets or livestock, for example withinveterinary settings.

This device may also be suitable as a research tool for multi-spectralimaging and monitoring of cancers in experimental animal models of humandiseases (e.g., wound and cancers). The device may be used to detectand/or image the presence of cancers and may be used to track tumorgrowth in animals models of cancer, particularly using fluorescent(e.g., in the visible and NIR wavelength ranges) protein transfectedtumor cell lines.

Image-Guidance

The device may also be useful for providing fluorescent image-guidance,for example in surgical procedures, even without the use of dyes ormarkers. Certain tissues and/or organs may have different fluorescentspectra (e.g., endogenous fluorescence) when viewed using the imagingdevice, or example under certain excitation light conditions.

FIG. 20 demonstrates the usefulness of the device for fluorescenceimaging-assisted surgery. With the aid of fluorescence imaging using thedevice, different organs of a mouse model may be more clearlydistinguishable than under white light. b, c and g show the mouse modelunder white light. a, d-f and h-j show the mouse model as imaged withthe device.

FIG. 20B shows an example of the use of the device for imaging smallanimal models. Here, the mouse dorsal skin-fold window chamber is imagedunder white light (a, c) and fluorescence (b, d). Note thehigh-resolution white light and fluorescence images obtained by thedevice. The feet and face appear bright red fluorescent due toendogenous autofluorescence from the cage bedding and food dustmaterials. (405 nm excitation; 490-550 nm and >600 nm emissionchannels).

Bioengineered Skin

Several bioengineered skin products or skin equivalents have becomeavailable commercially for the treatment of acute and chronic wounds, aswell as burn wounds. These have been developed and tested in humanwounds. Skin equivalents may contain living cells, such as fibroblastsor keratinocytes, or both, while others are made of acellular materialsor extracts of living cells (Phillips. J Dermatol Surg Oncol 1993;19(8): 794-800). The clinical effect of these constructs is 15-20%better than conventional ‘control’ therapy, but there is debate overwhat constitutes an appropriate control. Bioengineered skin may work bydelivering living cells which are known as a ‘smart material’ becausethey are capable of adapting to their environment. There is evidencethat some of these living constructs are able to release growth factorsand cytokines (Falanga et al. J Invest Dermatol 2002; 119(3): 653-60).Exogenous fluorescent molecular agents may be used in conjunction withsuch skin substitutes to determine completeness of engraftment as wellas biological response of the wound to the therapy. The healing offull-thickness skin defects may require extensive synthesis andremodeling of dermal and epidermal components. Fibroblasts play animportant role in this process and are being incorporated in the latestgeneration of artificial dermal substitutes.

The imaging device described here may be used to determine the fate offibroblasts seeded in skin substitute and the influence of the seededfibroblasts on cell migration and dermal substitute degradation aftertransplantation to wound site can be determined. Wounds may be treatedwith either dermal substitutes seeded with autologous fibroblasts oracellular substitutes. Seeded fibroblasts, labeled with a fluorescentcell marker, may then be detected in the wounds with fluorescenceimaging device and then quantitatively assessed using image analysis,for example as described above.

Polymer-Based Therapeutic Agents

There are a number of commercially available medical polymer productsmade for wound care. For example, Rimon Therapeutics produces Theramers™(www.rimontherapeutics.com) which are medical polymers that havebiological activity in and of themselves, without the use of drugs.Rimon Therapeutics produces the following wound care products, which canbe made to be uniquely fluorescent, when excited by 405 nm excitationlight: Angiogenic Theramer™, which induces new blood vessel development(i.e., angiogenesis) in wounds or other ischemic tissue; MI Theramer™,which inhibits the activity of matrix metalloproteases (MMPs), aubiquitous group of enzymes that are implicated in many conditions inwhich tissue is weakened or destroyed; AM Theramer™, a thermoplasticthat kills gram +ve and gram −ve bacteria without harming mammaliancells; and ThermaGel™, a polymer that changes from a liquid to a stronggel reversibly around body temperature. These can each be made to befluorescent by addition of fluorescent dyes or fluorescent nanoparticlesselected to be excited, for example, at 405 nm light with longerwavelength fluorescence emission.

By using the imaging device, the application of such fluorescent polymeragents may be guided by fluorescent imaging in real-time. This maypermit the Theramer agent to be accurately delivered/applied (e.g.,topically) to the wound site. Following application of the agent to thewound, the fluorescent imaging device may then be used to quantitativelydetermine the therapeutic effects of the Theramers on the wound as wellas track the biodistribution of these in the wound over time, in vivoand non-invasively. It may also be possible to add a molecular beacon,possibly having another fluorescent emission wavelength, to the MITheramer™ that can fluoresce in the presence of wound enzymes (e.g.,MMPs), and this may indicate in real-time the response of the wound tothe MI Theramer™. It may be possible to use one fluorescence emissionfor image-guided Theramer application to the wound site and anotherdifferent fluorescence emission for therapeutic response monitoring, andother fluorescence emissions for other measurements. The relativeeffectiveness of MMP inhibition and antimicrobial treatments may bedetermined simultaneously over time. Using image analysis, real-timecomparison of changes in fluorescence of these signals in the wound maybe possible. This adds a quantitative aspect to the device, and adds toits clinical usefulness.

It should be noted that other custom bio-safe fluorescence agents may beadded to the following materials which are currently used for woundcare. The fluorescent material may then be imaged and monitored usingthe device.

-   -   Moist Wound Dressings: This provides a moist conducive        environment for better healing rates as compared to traditional        dressings. The primary consumer base that manufacturers target        for these dressings is people over the age of 65 years,        suffering from chronic wounds such as pressure ulcers and venous        stasis ulcers. Those suffering from diabetes and as a result,        developed ulcers form a part of the target population.    -   Hydrogels: This adds moisture to dry wounds, creating a suitable        environment for faster healing. Their added feature is that they        may be used on infected wounds. These are also designed for dry        to lightly exudative wounds.    -   Hydrocolloid Dressings: Hydrocolloids seal the wound bed and        prevent loss of moisture. They form a gel upon absorbing        exudates to provide a moist healing environment. These are used        for light to moderately exudative wounds with no infection.    -   Alginate Dressings: These absorb wound exudates to form a gel        that provides a moist environment for healing. They are used        mainly for highly exudative wounds.    -   Foam Dressing: These absorb wound drainage and maintain a moist        wound surface, allowing an environment conducive for wound        healing. They are used on moderately exudative wounds.    -   Transparent Film Dressing: These are non-absorptive, but allow        moisture vapor permeability, thereby ensuring a moist wound        surface. They are intended for dry to lightly exudative wounds.        Examples include alginate foam transparent film dressings.    -   Antimicrobials: These provide antibacterial action to disinfect        the wound. Of particular interest is the use of nanocrystalline        silver dressings. The bio burden, particularly accumulated        proteases and toxins released by bacteria that hampers healing        and causes pain and exudation, is reduced significantly with the        extended release of silver.    -   Active Wound Dressings: These comprise highly evolved tissue        engineered products. Biomaterials and skin substitutes fall        under this category; these are composed entirely of biopolymers        such as hyaluronic acid and collagen or biopolymers in        conjunction with synthetic polymers like nylon. These dressings        actively promote wound healing by interacting either directly or        indirectly with the wound tissues. Skin substitutes are        bioengineered devices that impersonate the structure and        function of the skin.    -   Hyaluronic Acid: This is a natural component of the extra        cellular matrix, and plays a significant role in the formation        of granular tissue, re-epithelialization and remodeling. It        provides hydration to the skin and acts as an absorbent.

Other wound care products that may be imaged using the disclosed deviceinclude Theramers, silver-containing gels (e.g., hydrogels), artificialskin, ADD stem cells, anti-matrix metalloproteinases, and hyaluronicacid. Fluorescent agents may be added to other products to allow forimaging using the device. In some cases, the products may already beluminescent and may not require the addition of fluorescent agents.

The device may be used also to monitor the effects of such treatmentsover time.

Application for Food Products

The imaging device may also be useful for monitoring food products(e.g., meat products) for contamination. This may be useful, forexample, in food/animal product preparation in the meat, poultry, dairy,fish, and agricultural industries. The device may be used as part of anintegrated multi-disciplinary approach to analytical laboratory serviceswithin this sector, which may provide capabilities including image-baseddetection of contamination and guidance for obtaining samples fortesting. The device may be used for real-time detection, identificationand monitoring of level of bacterial and other microbial meatcontamination/adulteration of food products. It may be used forbacterial contamination tracking in the food processing plantenvironment, and thus may provide an image-based method for determiningfood safety and quality. In embodiments where the device is hand-held,compact and portable, the imaging device may be useful in foodpreparation areas to determine safety of food products frombacterial/microbial contamination. The device may also be used forrelatively rapid detection and analysis of bacteria/microbes in meatsamples (and on preparation surfaces) collected or sampled, for exampleas part of food-safety and quality regulated inspection process, duringprocessing and in finished food products. This device may be used in themeat, horticulture and aquaculture industries in implementing foodsafety inspection/detection procedures that meet the requirements forfood safety and quality. The device may be used to detect foodcontaminants, for example contaminants found in the meat, poultry, dairyand fish industries. This technology may be useful for as a fecalcontaminant detection system, since fecal bacteria produce porphyrinswhich may be readily detected by the device.

Detection and accurate identification of foodborne pathogens, such asListeria monocytogenes (LM), in food samples and processing lines may becritical both for ensuring food quality assurance and tracing ofbacterial pathogen outbreaks within the food supply. Current detectionmethods employed in food production and processing facilities typicallyrely on multiple random surface sampling of equipment (e.g., swabbing),and subsequent molecular-based diagnostic assays (e.g., real-timepolymerase chain reaction, RT-PCR) which may provide quantitativeconfirmation of the presence of LM, typically within 24-72 h. However,given time and cost restraints, typically only randomized selected zonesof a given food production facility are tested for pathogencontamination at a time, and the significant potential of under-samplingduring the “first pass” surface swabbing of equipment may result inundetected pathogens causing catastrophic health and economicconsequences. In addition, the inability to i) rapidly sample allsurface areas during the “first pass” swabbing to identify areas withhigh infection probability , ii) to visually document this initialscreening process (e.g. no imaging methods available to date), iii) thedelay in obtaining laboratory results, iv) the high-costs associatedwith current methods, and v) more importantly, the potential of missingdeadly pathogen infections have prompted efforts to improve the earlyand accurate detection of food-born pathogens cost-effectively.

The device may be useful in providing a relatively rapid and accurateway of detecting such pathogens. The device may be used with an assay ofa multi-coloured fluorescence probe ‘cocktail’ (e.g., a combination oftwo or more contrast agents) which may unequivocally identify (and maymake visible) only viable Listeria monocytogenes from other Listeriaspecies using highly-specific gene probe technology. This may allowspecific detection of living LM in real-time, potentially minimizing theneed for standard time-consuming enrichment methods. This method mayalso be expanded to include detection of other pathogens of interest,including Enterobacter sakazakii, Campylobacter species (C. coli, C.jejuni and C. lari), coliform bacteria and bacteria of the species E.coli (including lactose- and indol-negative Escherichia coli-strains),Salmonella, all bacteria belonging to the species Staphylococcus aureusand separately all bacteria belonging to the genus Staphylococcus, andPseudomonas aeruginosa. Other bacteria may be detectable by selecting asuitable probe or combination of probes. For example a combination oftwo or more contrast agents may be designed to be specific to a certainbacteria, and may result in a unique detectable fluorescent signaturewhen imaged using the imaging device.

The imaging device may be used (e.g., when combined with appliedexogenous bacteria-specific contrast agents, including a multi-targetedprobe or a combination of probes) for relatively rapid “first pass”screening of food-preparation and handling surfaces for targetedswabbing and microbiological testing. This device may allow relativelyrapid image-based surveillance of any surface of equipment and foodproducts and may capture the fluorescence signature of food-bornebacteria/pathogens in real-time. The device may be used in combinationwith, for example, an assay of a multi-coloured fluorescence probe‘cocktail’ (and combinations thereof) which may unequivocally identify(and may make visible) only viable Listeria monocytogenes from otherListeria species using highly-specific gene probe technology, asdescribed above. Such a probe ‘cocktail’ may be designed to specificallytarget certain pathogens based on a specific combination of probes knownto be sensitive to such pathogens, and known to give a signaturefluorescence response. In addition to detection of such pathogens, thedevice may allow for the presence and/or location of different strainsto be differentiated, based on their different signature fluorescenceresponse.

FIG. 26 shows an example of the use of the imaging device for real-timeexamination of meat products in the food supply. Here, a) white lightand b) corresponding autofluorescence imaging of a piece of pork meatshows the difference between various tissues including bone and tendon(white arrow), fat, and muscle. c) White light and b) correspondingautofluorescence imaging of a ‘cut-on edge’ of bone, where cartilage(blue arrow) appears bright green under fluorescence light due tocollagen autofluorescence, while various types of inner bone tissuesincluding bone marrow (red arrow) can be differentiated usingfluorescence. The latter observation may additionally suggest the use ofthe handheld optical imaging device for real-time fluorescenceimage-guidance during orthopedic surgery in human and veterinarypatients, as discussed above. (405 nm excitation, 500-550 nm emission(green), >600 nm emission (red)) .

FIG. 27 shows another example of the use of the imaging device forreal-time examination of meat products in the food supply. Here, a)white light and b) corresponding autofluorescence imaging of a piece ofpork meat that has been maintained for 2 days at 37° C. Autofluorescenceimaging shows the presence of a mixed bacterial contamination on themeat surface (red fluorescence areas; yellow arrows) including, forexample, Staphylococcus aureus and E. coli. (405 nm excitation, 500-550nm emission (green), >600 nm emission (red)).

Surface Contamination

The imaging device may be useful for detection of surface contamination,such as for detection of ‘surface bacterial contamination’ in healthcare settings. This device may be used for detecting and imaging of thepresence of bacteria/microbes and other pathogens on a variety ofsurfaces/materials/instruments (in particular those related to surgery)in hospitals, chronic care facilities, and old age homes, wherecontamination is the leading source of infection. The device may be usedin conjunction with standard detection, identification and enumerationof indicator organisms and pathogens strategies.

FIG. 28 shows an example of the use of the imaging device for real-timeexamination of soil and algae samples, in an example of environmentalsampling/detection of contaminants. A) White light and b) correspondingautofluorescence images of a Petri dish containing a soil and mineralsample. c) An example of the imaging device used to detect fluorescentsoil contaminants/hazardous materials. Here, for example, afluorescein-labeled fluid was added to the soil prior to fluorescenceimaging to illustrate the potential use of the imaging device fordetection and monitoring of environmental pollutants and contaminants.d) An example of the imaging device used to obtain white light and e)autofluorescence images of a green algae culture grown under laboratoryconditions, illustrating the potential utility of the imaging device forreal-time fluorescence image-based monitoring of water conditions (e.g.,drinking water purification/safety testing, or algae growth inlarge-scale production plants). As an example of the imaging device usedto detect disease in plants, f) shows a white light image of a commonhouse plant while g) shows the corresponding autofluorescence image of afungal infection appearing bright green (yellow arrows) affecting theplants leaves, compared to healthy leaf tissue which appears brightreddish-brown. (405 nm excitation, 500-550 nm emission (green), >600 nmemission (red)). Thus, the device may be useful for imaging plantmaterials.

FIG. 28B shows an example of the use of the imaging device used fordetection of white light-occult contamination of biological fluids inpublic and private environments. a) White light and b), c) correspondingautofluorescence of the biological fluids contaminating a toilet seatand a bathroom vanity countertop. These data suggest that the imagingdevice may be used for detecting surface contamination by potentiallyhazardous biological/infectious fluids/samples for image-guided targetedsampling, cleaning or monitoring. (405 nm excitation, 500-550 nmemission (green), >600 nm emission (red)) .

FIG. 28C shows an example of the use of the device for detection ofbacterial contamination of surgical instrumentation (b; green arrow)using fluorescence imaging. (405 nm excitation; 490-550 nm and >600 nmemission channels).

Forensic Uses

The use of the imaging device to image surface contaminants and targetsmay be useful in forensic applications. For example, the device may beuseful for forensic detection of latent finger prints and biologicalfluids on non-biological surfaces. The device may offer a relativelyinexpensive, compact and portable means of digitally imaging (e.g., withwhite light, fluorescence and/or reflectance) latent finger prints andbiological fluids, and other substances of forensic interest. The formermay be made fluorescent using commercially available finger printfluorescence dyes, and the latter may be detected either usingautofluorescence of the fluids or exogenously applied ‘targeted’fluorescent dye agents (such as Luminol). Images may be recordeddigitally. The device may also be used during autopsy procedures todetect bruising

FIG. 29 shows an example of the use of the imaging device for real-timefluorescence detection of liquid leaks using a exogenous fluorescentleak-tracer dye. a) White light image of a typical faucet, b)corresponding fluorescence image (showing the presence of the leakingfluid (with fluorescence dye added), and composite image of white lightand fluorescence. Note that the leak (in this example, water) is notvisible under white light, but is easily detected using fluorescence.These data suggest the imaging device may be useful for relatively rapidimage-based tracing and detection of leaks of liquids/fluids (405 nmexcitation, 500-550 nm emission (green), >600 nm emission (red)).

FIG. 30 shows an example of the use of the imaging device for real-timefluorescence detection of surface contaminants). a) White light image ofa typical laboratory bench surface and b) an area that is to be imagedusing the imaging device. c) Fluorescence imaging may be used to detectcontaminants that are not easily visualized under white light (a, b).

The imaging device may also be used to detect latent fingerprints, forexample by using a fluorescent dye to enhance the finger print ridges ona table surface. This may be done, for example, by including fluorescentdye combined with superglue (e.g., cyanoacrylate) to develop fingerprintcontrast against background surfaces. Far-red and near-infraredfluorescent dyes may be used to reduce the potential of backgroundautofluorescent. These data suggest the use of the imaging device forrelatively rapid image-based detection of non-biological and biologicalcontaminants as well as fingerprints, for example, in forensicapplications. (405 nm excitation, 500-550 nm emission (green), >600 nmemission (red)).

The device may also be useful in anti-counterfeit applications. FIG. 31shows an example of the imaging device being used for imaging of commoncurrency (in this example, a Canadian $20 bill) under a) white light andb, c) autofluorescence modes. Invisible under white light (a), specialanti-counterfeiting measures may be seen under fluorescence: i.e.,embedded fluorescence fibers (b) and embedded watermarking of bank notes(c) can be spectrally distinguished (arrows). These data suggest thatthe device may be used for anti-counterfeiting purposes. (405 nmexcitation, 500-550 nm emission (green), >600 nm emission (red)).

Cataloguing

The imaging device may be allow for fluorescent-based cataloguing ofanimals, such as laboratory animals. FIG. 32 shows an example of the useof the imaging device for real-time fluorescence detection ofidentification “barcode” tagging for laboratory animals. The figureshows a) white light image of a typical laboratory rat and b) afluorescence image of the rat tagged with a fluorescent barcode. The useof multiple fluorescent dyes/colors in combination with barcodepatterns/bars may be used for ‘multiplexed cataloguing’ of animals, forexample for longitudinal research studies. These data suggest the use ofthe imaging device for relatively rapid high-throughput image-basedbarcode cataloguing of laboratory animals for use in c)“pathogen-containment” animal colonies in research laboratories and foranimal genotyping (e.g. transgenic animals, inset in c), for examples.(405 nm excitation, 500-550 nm emission (green), >600 nm emission(red)). The device may also be used for imaging of fluorescence-basedbarcoding or other coding systems in other applications, such asinventory tracking and point-of-sale tracking.

Kits for Device

The imaging device may be provided in a kit, for example including thedevice and a fluorescing contrast agent. The contrast agent may be anyone or more of those described above. For example, the contrast agentmay be for labeling a biomarker in a wound, where the kit is for woundmonitoring applications.

FIG. 33 shows an example of a kit including the imaging device. a) showsthe handle and the touch-sensitive viewing screen, and b) shows externalhousing and excitation light sources. The imaging device may be used toscan the body surface of both human and veterinary patients forimage-based wound assessment, or for non-wound imaging applications. Thedevice and any accessories (e.g., electrical/battery power supplies),potential exogenous fluorescence contrast agents, etc.) may beconveniently placed into hard-case containers for transport withinclinical and non-clinical environments (including remote sites, homecare and research laboratory settings).

Cosmetic or Dermatology Uses

The imaging device may also be used for imaging cosmetic ordermatological products.

FIG. 34 shows an example of the use of the device for imaging ofcosmetic products. For example, four commercially available cosmeticcreams are shown under a) white light and b) fluorescence imaging modes,showing fluorescence contrast between the creams and the backgroundskin. These data illustrate the potential use of the handheld imagingdevice for use in imaging the presence and potential biological effectsof cosmetic (e.g. rehydration of skin, collagen remodeling, repairingsunburn damage, skin exfoliation) and/or dermatological agents or drugs(405 nm excitation; 490-550 nm and >600 nm emission channels)).

The imaging device may be used in white light and fluorescence modes toimprove the administration of these treatments as well as monitor theireffectiveness over time non-invasively and quantitatively. The devicemay be used in combination with other imaging modalities, for examplethermal imaging methods, among others.

This device may also be used to test anti-bacterial, antibiotic, ordisinfectant agents. Fluorescence imaging provided by this device may beused, for example in combination with white light imaging, toquantitatively detect the effectiveness of pharmaceutical treatments inbacterial cultures and other model systems, during drug discovery,optimization, and evaluation, for example for wound treatment.

All examples and embodiments described herein are for the purpose ofillustration only and are not intended to be limiting. A person skilledin the art would understand that other variations are possible. Allreferences mentioned are hereby incorporated by reference in theirentirety.

We claim:
 1. A method of screening for contamination during foodproduction, comprising: applying an exogenous, bacteria-specificcontrast agent to a surface to be screened, wherein the surface to bescreened is one or more of a food product, a food-preparation surface, afood-handling surface, and a food-equipment surface; illuminating thesurface on which the contrast agent has been applied with excitationlight emitted by at least one light excitation light source of ahandheld imaging device; filtering, with at least one optical filter ofthe handheld device, optical signals responsive to illumination of thesurface including the contrast agent with the excitation light, the atleast one optical filter being configured to enable optical signalshaving a wavelength corresponding to bacterial fluorescence to passthrough the filter; collecting bacterial fluorescence data regarding theilluminated surface with an image sensor of the handheld imaging device,wherein the collected bacterial fluorescence data comprises at least oneimage containing data relating to exogenous fluorescence of bacteriaresponsive to illumination of the surface; and in real-time, based onthe collected bacterial fluorescence data, determining the presenceand/or location of bacteria on the illuminated surface.
 2. The method ofclaim 1, wherein applying an exogenous, bacteria-specific contrast agentto the surface to be screened includes applying a mixture of two or morecontrast agents.
 3. The method of claim 1, wherein the exogenous,bacteria-specific contrast agent is configured to make visible onlyspecific strains of bacteria.
 4. The method of claim 1, wherein theexogenous, bacteria-specific contrast agent is configured to makevisible only viable Listeria monocytogenes.
 5. The method of claim 1,wherein the exogenous, bacteria-specific contrast agent is configured tomake visible one or more of Listeria monocytogenes, Enterobactersakazakii, Campylobacter coli, Campylobacter jejuni, Campylobacter lari,coliform bacteria, all bacteria of the E. coli species, Salmonella, allbacteria of the Staphylococcus aureus species, all bacteria of theStaphylococcus genus, and Pseudomonas aeruginosa.
 6. The method of claim1, further comprising displaying an image of bacteria detected on theilluminated surface on a display of the handheld imaging device or on adisplay configured to receive transmissions from the handheld imagingdevice.
 7. The method of claim 1, further comprising, when bacteria aredetermined to be present on the illuminated surface, identifying one ormore strains of bacteria present.
 8. The method of claim 1, furthercomprising, when bacteria are determined to be present on theilluminated surface, differentiating between strains of bacteria presenton the illuminated surface.
 9. A method for fluorescence-based imagingof a target to detect contamination comprising: labelling a selectedbiomarker at the target with at least one fluorescing contrast agent;illuminating the target with excitation light emitted by an excitationlight source and having at least one wavelength or wavelength bandcausing at least the selected biomarker to fluoresce; detectingfluorescence emissions of at least the selected biomarker with an imagedetector of a handheld imaging device; and determining the presence,location, and/or quantity of contamination on the illuminated targetbased on the detected fluorescence emissions of at least the selectedbiomarker, wherein the target is selected from the group consisting of afood product, a food-preparation surface, a food-handling surface, afood packaging surface, an area in a food processing plant, an area in afood production facility, and a food-equipment surface.
 10. The methodof claim 9, further comprising comparing fluorescence emission band(s)of the at least one biomarker to a predetermined look-up table offluorescence emission spectra and/or fluorescence emission band(s) ofbiomarkers.
 11. The method of claim 9, wherein labelling the selectedbiomarker at the target comprises labelling the selected biomarker witha combination of two or more contrast agents, wherein the combination isspecific to the selected biomarker.
 12. The method of claim 9, whereinlabelling the selected biomarker at the target comprises labelling theselected biomarker with an exogenous, bacteria-specific contrast agent.13. The method of claim 12, wherein the exogenous, bacteria-specificcontrast agent is configured to make visible one or more of Listeriamonocytogenes, Enterobacter sakazakii, Campylobacter coli, Campylobacterjejuni, Campylobacter lari, coliform bacteria, all bacteria of the E.coli species, Salmonella, all bacteria of the Staphylococcus aureusspecies, all bacteria of the Staphylococcus genus, and Pseudomonasaeruginosa.
 14. The method of claim 9, wherein determining the presence,location, and/or quantity of contamination on the illuminated targetincludes detecting the presence, location, and/or quantity of abacterial strain based on the fluorescence emissions of at least theselected biomarker.
 15. The method of claim 14, wherein the bacterialstrain is at least one selected from the group consisting of:Staphylococcus bacteria, Staphylococcus aureus, Pseudomonas aeruginosa,Listeria monocytogenes, Enterobacter sakazakii, Campylobacter species ofbacteria, coliform bacteria, Escherichia coli bacteria,Propionibacterium acnes and Salmonella.
 16. The method of claim 14,wherein detecting the presence, location, and/or quantity of a bacterialstrain based on the fluorescence emissions of at least the selectedbiomarker includes differentiating the presence, location and/orquantity of two or more different bacterial strains based on thefluorescence emissions of at least the selected biomarker.
 17. Themethod of claim 16, wherein the different bacterial strains includeStaphylococcus aureus and Pseudomonas aeruginosa, and the differentbacterial strains are differentiated based on their fluorescenceemission signatures.
 18. The method of claim 9, wherein the selectedbiomarker is chosen from the group consisting of: bacteria, fungi,yeast, spores, virus, microbes, parasites, connective tissues, tissuecomponents, exudates, pH, blood vessels, reduced nicotinamide adeninedinucleotide (NADH), flavin adenine dinucleotide (FAD), microorganisms,vascular endothelial growth factor (VEGF), endothelial growth factor(EGF), epithelial growth factor, epithelial cell membrane antigen(ECMA), hypoxia inducible factor (HIF-1), carbonic anhydrase IX (CAIX),laminin, fibrin, fibronectin, fibroblast growth factor, transforminggrowth factors (TGF), fibroblast activation protein (FAP), tissueinhibitors of metalloproteinases (TIMPs), nitric oxide synthase (NOS),inducible and endothelial NOS, lysosomes in cells, macrophages,neutrophils, lymphocytes, hepatocyte growth factor (HGF),anti-neuropeptides, neutral endopeptidase (NEP), granulocyte-macrophagecolony stimulating factor (GMCSF), neutrophil elastases, cathepsins,arginases, fibroblasts, endothelial cells and keratinocytes,keratinocyte growth factor (KGF), macrophage inflammatory protein-2(MIP-2), macrophage inflammatory protein-2 (MIP-2), and macrophagechemoattractant protein-1 (MCP-1), polymorphonuclear neutrophils (PMN),macrophages, myofibroblasts, interleukin-1 (IL-1), tumour necrosisfactor (TNF), nitric oxide (NO), c-myc, beta-catenin, endothelialprogenitor cells (EPCs), matrix metalloproteinases (MMPs) and MMPinhibitors.
 19. The method of claim 9, wherein illuminating the targetwith a light source emitting excitation light having at least onewavelength or wavelength band causing at least the selected biomarker tofluoresce comprises illuminating the target with a light source emittinglight having a wavelength between about 400 nm and about 450 nm.
 20. Themethod of claim 9, wherein detecting fluorescence emissions of at leastthe selected biomarker with the image detector of the handheld imagingdevice includes filtering emissions responsive to illumination of thetarget with one or more filters configured to permit optical signalshaving wavelengths corresponding to bacterial fluorescence to passthrough the one or more filters to the image detector of the handhelddevice.
 21. The method of claim 20, wherein the wavelengthscorresponding to bacterial fluorescence include wavelengths of about 490nm to about 550 nm and/or wavelengths above about 600 nm.
 22. The methodof claim 9, wherein determining the presence, location, and/or quantityof contamination on the illuminated target comprises displaying thedetected fluorescence emissions on a display of the handheld imagingdevice or a display configured to receive transmissions from thehandheld device.
 23. The method of claim 9, further comprisingdisplaying an image of a location and/or biodistribution of bacteria,microorganisms, selected biomarker(s), and/or pathogens detected on theilluminated target on a display of the handheld device or a displayconfigured to receive transmissions from the handheld device.